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Comparison of anti-p53 antibodies in immunoblotting
Biochemical and Biophysical Research Communications 293 (2002) 850–856 www.academicpress.com
Comparison of anti-p53 antibodies in immunoblotting Miia Turpeinen, Raisa Serpi, Mika Rahkolin, and Kirsi V€ ah€ akangas* Department of Pharmacology and Toxicology, P.O. Box 5000, FIN-90014 University of Oulu, Oulu, Finland Received 25 March 2002
Abstract Some of the most important tools to study p53 protein are various anti-p53 antibodies and immunological methods based on antibody–antigen reactions. Critical comments on the specificity and sensitivity of anti-p53 antibodies have been published. Four monoclonal and two polyclonal anti-p53 antibodies, four of them from two different sources, were compared for their ability to detect in immunoblotting the benzo(a)pyrene-induced p53 from C57BL/6 mouse skin and MCF-7 human breast carcinoma cells. Multiple extra bands were seen with most antibodies. A theoretical comparison of the equivalent epitopes of p53 homologues with the known epitopes of p53 antibodies indicated that the extra bands seen with most antibodies are not due to cross-reactivity with these homologues. A careful adjustment of antibody dilutions is needed for each application utilizing commercial p53 antibodies, regardless of the recommendations of the supplier. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Immunoblotting; Anti-p53 antibodies; MCF-7 cell line; Mouse skin; Epitopes; p53 Homologues
p53 is one of the most studied proteins because of its crucial role in the cell cycle and the fact that over 50% of all cancers contain a mutation in TP53 gene [1–3]. As a transcription factor p53 induces cell cycle arrest, DNAdamage repair, and apoptosis by increasing the expression of a number of other genes such as p21/WAFl, BAX, GADD45, TFIID, TFIIH, MDM2, and ARF [4,5]. Human p53 protein consists of 393 and mouse p53 protein of 390 amino acid residues. p53 protein can be divided into five different functional and structural domains [4]. The N-terminal end of p53 acts as a transactivation area (amino acids 1–42), which binds at the promoter regions the regulatory sequences of its target genes. Carboxy-terminal domain of p53 (amino acid residues 360–393) regulates the activity of p53 protein and binds DNA non-specifically. The highly conserved central core region between amino acids 100 and 300 binds DNA in a sequence-specific manner [6,7]. The majority of known p53 mutations are located within this
*
Corresponding author. Permanent address: Department of Pharmacology and Toxicology, University of Kuopio, P.O. Box 1627, FIN70211 Kuopio, Finland. Fax: +358-17-162-424/+358-8-537-5247. E-mail addresses: kirsi.vahakangas@oulu.fi, kirsi.vahakangas@ uku.fi (K. V€ ah€ akangas).
core region of the protein [2,3]. Most of the antibody epitopes as well as phosphorylation and acetylation sites are found at the ends of the p53 protein [8,9]. Posttranslational modifications have importance in stabilization and activation of a p53 protein [10]. Immunoblotting has been suggested ‘‘as the gold standard for demonstrating p53 expression’’ [11]. However, many anti-p53 antibodies used in immunoblotting have not performed according to the expectations. Critical comments on p53 antibodies and unidentified extra bands have been reported [12–15]. For a comparison of the results between laboratories there is clearly a need for more intra- and inter-laboratory validation of the immunomethods. We have compared in immunoblotting six different anti-p53 antibodies, suitable for this purpose according to the supplier. The detectability of p53 protein obtained from MCF-7 cells and mouse skin cells treated by benzo(a)pyrene was studied. We have shown before that p53 protein is inducible by benzo(a)pyrene in these models [16–19]. Materials and methods Antibodies. Anti-p53 antibodies PAb 421, CM1, DO7, PAb 1801, and PAb 240 were evaluated for their ability to detect p53 from MCF-7 human breast cancer cells. PAb 240, PAb 421, and CM5
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 2 9 8 - X
M. Turpeinen et al. / Biochemical and Biophysical Research Communications 293 (2002) 850–856 antibodies were used for p53 from C57BL/6 mouse skin cells (Fig. 1, Table 1). All of the commercial antibodies are recommended, among other applications, also for immunoblotting. CM5 and one of the two CM1 antibodies used were original antibodies from Prof. David Lane. Other antibodies were from commercial suppliers. All antibodies were titrated to reach the optimal background and visibility of the p53 band. Secondary antibody used against mouse monoclonal antibodies DO7, PAb 1801, PAb 240, and PAb 421 was a polyclonal sheep anti-mouse IgG conjugated with horseradish per-
851
oxidase (Batch 144832, Amersham Life Science, Buckinghamshire, UK). The secondary antibody against antibodies CM1 and CM5 was a polyclonal goat anti-rabbit antibody conjugated with horseradish peroxidase (Lot# B16624, Oncogene Research Products, Cambridge, MA). Mice. Male C57BL/6 mice (12–15 weeks of age) were maintained in plastic cages at a constant temperature of 25 °C with a 12 h lightdark-cycle and had free access to food (Standard rodent pellets, Special Diets Services, Essex, England) and water. The backs of the mice were
Fig. 1. Epitopes for anti-p53 antibodies used in this study. Functional domains of p53 protein are indicated by boxes I transactivation domain, II proline-rich region, III specific DNA-binding domain, IV oligomerization domain, V basic C-terminal domain. Amino acid numbers for both functional areas and antibody epitopes are indicated.
Table 1 Antibodies used in this study Primary antibody
Manufacturer
Type of antibody
Specificity
Used working dilution
Recommended working dilution
Reference
MCF-7 DO7 DO7 PAb 1801 PAb 1801 PAb 421
NCL ST ORP SCB ORP
Mouse Mouse Mouse Mouse Mouse
1:2000 1:1000 1:1000 1:500 1:100
1:1000–1:5000 1:1000 2:5 lg=ml (1:400) – 10 lg=ml (1:100)
[34] [34] [35] [34] [36]
PAb 240
SCB
Mouse monoclonal
1:200
–
[34]
PAb 240 CM1 CM1
ORP NCL
Mouse monoclonal Rabbit polyclonal Rabbit polyclonal
Human wt/mut Human wt/mut Human wt/mut Human Human, rat, mouse, monkey rabbit Human, rat, mouse, avian, bovine Human, mouse Human wt/mut Human
1:200 1:16,000 1:32,000
5 lg=ml (1:200) 1:500–1:1000 –
[37] [38] [38]
1:2000
10 lg=ml (1:100)
[36]
1:4000 1:4000 1:2000
– 5 lg=ml (1:200) –
[34] [37] [39]
Mouse skin PAb 421 PAb 240 PAb 240 CMS
a
monoclonal monoclonal monoclonal monoclonal monoclonal
ORP
Mouse monoclonal
SCB ORP
Mouse monoclonal Mouse monoclonal Rabbit polyclonal
a
Human, rat, mouse, monkey rabbit Human, mouse Human, mouse Mouse
NCL, Novocastra Laboratories (UK); ST, Serotec (UK); ORP, Oncogene Research Products (MA, USA); SCB, Santa Cruz Biotechnology (CA, USA). a Gift from Prof. David Lane (Dundee, UK).
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shaved three days before the treatments and only mice with fur in a non-growing phase were selected. Five hundred micrograms of benzo(a)pyrene (BP) (Sigma, St. Louis, MO) in 100 ll of acetone was applied on the shaved backs of the mice. Control animals were treated with 100 ll of acetone. Skin samples were collected 24 h after the treatment when the p53 response was strongest [16,17]. For immunoblotting analysis the treated mouse skin was removed and subcutaneous fat was removed on ice. The mouse skin samples were homogenized on ice for 10 min in 1.5 ml nuclear lysis buffer (20% glycerol, 20 mM HEPES, 500 mM NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 1 mM DTT, 0.1% NP40, 100 lg=ml PMSF, 1 lg=ml aprotinin, 1 lg=ml pepstatin A, and 1 lg=ml antipain). The samples were incubated for 20 min on ice and then centrifugated for 15 min at 12,000 rpm at +4 °C. The supernatant (whole cell extract) was collected and the samples were maintained at )70 °C until needed. MCF-7 cells. MCF-7 cells, which express wild type p53 protein [18], were cultured at 37 °C in an atmosphere of 95% humified air and 5% CO2 . The used RPMI 1640 medium contained non-essential amino acids, 10% fetal bovine serum, 1 mM streptomycin, 1 mM penicillin, 2 mM glutamine (all from Gibco, Paisley, UK), 1 lg=ml estradiol (Sigma, St. Louis, MO), and 1 lg=ml insulin (Novo Nordisk, Bagsværd, Danmark). To increase the amount of p53 protein subconfluent cell cultures were treated with either 1 lM or 5 lM benzo(a)pyrene (BP) (Sigma, St. Louis, MO). BP was diluted in acetone and 50 ll of the dilution was added to the medium for 48 h [18]. The control cells were treated with acetone. For immunoblotting, MCF-7 cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in 150 ll nuclear lysis buffer. The samples were incubated for 20 min on ice and then centrifuged for 15 min at 12,000 rpm at +4 °C. The supernatant (whole cell extract) was collected and the samples were maintained at )70 °C until needed. Immunoblotting (Western blotting). Thirty micrograms of protein (50 lg of MCF-7-samples for PAb 240 and PAb 421) was heated for 4 min at 96 °C in Laemmli buffer and separated electrophoretically at 200 V (Bio-Rad Power Pac 200, Bio Rad, Hercules, CA) in a 10% polyacrylamide gel containing SDS. Polyvinylidene difluoride (PVDF) filter (Immobilon P, 0.45 lm pore size, Millipore, Bedford, MA) was treated according to the instructions of the manufacturer and the proteins were transferred to a filter by electroblotting for 1 h at 100 V. To avoid unspecific binding, the filters were blocked overnight at +4 °C in Tris-buffered saline (TBS) containing 5% non-fat cow milk powder. The filters were incubated in a primary antibody at room temperature for 1 h, washed five times with TBS containing 0.05% Tween 20, incubated for 1 h in a secondary antibody at room temperature and washed again thoroughly with TBS–Tween. Enhanced chemiluminescence system (ECL+, Amersham Life Science, Buckinghamshire, UK) was used according to the instructions of the manufacturer. The size of a band was defined by Rainbow Coloured Protein Molecular Weight Marker (Amersham Life Science, Buckinghamshire, UK) analysed in parallel with the samples. Immunoblotting was repeated from two to nine times depending on the antibody and only reproducible results are reported. For comparisons, the same three samples of MCF-7 and the same two samples from mouse skin were used throughout the experiments. Hexosaminidase treatment to digest the possible sugar residues in the blots was done after the blocking of unspecific binding in milk according to Shaw et al. [20] and Jackson and Tjian [21]. Briefly, the filters were rinsed quickly with 50 mM sodium citrate, pH 5.0 (Sigma, St. Louis, MO) and then incubated at 37 °C for 3, 6, 12, or 24 h with 2.5 units of b-N-acetylglucosaminidase (Sigma, St. Louis, MO) in 5 ml sodium citrate. The blots were then extensively washed with TBS– Tween, re-equilibrated in 5% TBS–milk and then blotted with the primary antibody as above. Computer analysis of the protein sequences. Protein sequences were obtained by using SWISS-PROT/TrEMBL—database (http:// www.expasy.ch/cgi-bin/sprot-search-de). All sequences were compared against the theoretical epitope area of each antibody by using SIM-
Alignment analysis tool for protein sequences (http://www.expasy.ch/ tools/sim-prot.html). The sequences used were for mouse O70366 (p53), O88897 (p63 TA c), O88898 (p63 TA a), O88899 (p63 DN c), O89097 (p63 DN a), and Q9WUJO (p73 fragment) and for human P04637 (p53), Q07065 (p63), and O15350 (p73). Program was used by default parameters (Comparison matrix BLOSUM62, gap open penalty 12, gap extension penalty 4). Comparison of the sequences is expressed as an SIM-score, which is SIM-program’s best score for alignment of two sequences. For instance, the score for the amino acid sequence of DO7 epitope and human p53 is 34 which is reached when all six amino acids of the epitope are in correct order and detectable in the protein. To compare the theoretical possibilities of different proteins to be detectable by a certain antibody, a percentage of the SIMscore from a perfect match (human p53) was calculated (SAHP53).
Results The dilutions of the antibodies recommended by the suppliers were not the best in all cases for the ECL+ based detection of p53 (Table 1). In mouse skin cells, all the antibodies used (PAb 240 from two commercial sources, PAb 421 and CM5) detected p53 protein and its increase by BP treatment (Fig. 2). However, the intensity of the bands and the strength of the induction differed depending on the antibody. CM5, which did not detect any band at 53 kDa in the untreated skin, and PAb 240 from SCB showed the clearest indication of p53 response, while with PAb 421 the induction was barely detectable. PAb 421 and both PAb 240 antibodies detected a protein slightly larger in size than the band detected by CM5.
Fig. 2. Immunoblots from untreated (lane 1) and benzo(a)pyrenetreated (lane 2: 500 lg for 24 h) skin from C57BL/6 mice. (a) CM5; (b) PAb 421 (ORP); (c) PAb 240 (ORP) and (d) PAb 240 (SCB). The used working dilution for each antibody is presented in Table 1. Thirty micrograms of protein was used for all antibodies. p53-Band is indicated with an arrow.
M. Turpeinen et al. / Biochemical and Biophysical Research Communications 293 (2002) 850–856
In skin cells from in vivo-treated mice all four antibodies showed a strong band of approximately 66 kDa. This band was similar in intensity to BP-treated and control samples. CM5 also showed a smaller band of 46 kDa and PAb 240, and PAb 421 a weak band of
853
60 kDa. There was no difference in the intensities of these bands between treated and control samples. In MCF-7 samples the p53 band was detectable when antibodies CM1, PAb 1801, and DO7 were used (Fig. 3). Multiple bands with the polyclonal antibody CM1 (from
Fig. 3. Immunoblots from untreated (lane 1) and benzo(a)pyrene-treated (lane 2: 1 lM for 48 h; lane 3: 5 lM for 48 h) MCF-7 cells: (a) DO7 (NCL); (b) DO7 (ST); (c) PAb 1801 (ORP); (d) PAb 1801 (SCB); (e) CM1 (NCL); (f) CM1 (a gift from Prof. David Lane, Dundee, UK); (g) PAb 240 (ORP); (h) PAb 240 (SCB); (i) PAb 421 (ORP). The used working dilution for each antibody is presented in Table 1. Thirty micrograms of protein was used for a–f and 50 lg for g–i. The site of 53 kDa band is indicated with an arrow.
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97 kDa. Computer analysis of PAb 421 showed that the site of the epitope in p53-related proteins was less than 40% similar with the epitope of p53. Thus the probability that PAb 421 detects other than p53 protein is small.
NCL and Prof. David Lane) are easily explained because each polyclonal antibody is a serum of the immunized animal and thus contains antibodies against many epitopes. With both of the CM1 antibodies the main bands were at about 35, 48, 53, 63, and 66 kDa. Some weak bands between these regions were also detectable through the whole area. The benzo(a)pyrene induction was also seen with CM1 after 5 lM BP treatment, although not very strongly. Both DO7 antibodies (ST, NCL) gave one sharp band in MCF-7 cells at 53 kDa while PAb 1801 antibodies showed bands at 53 and 46 kDa and a weak band at about 63 kDa. PAb 1801 (ORP) detected also a band at 66 kDa. The dosedependent p53 response was most obvious with the DO7 antibody. With the DO7 from NCL the induction of p53 protein was seen already with 1 lM BP, while the DO7 from ST showed an increase only with 5 lM BP. Both PAb 1801 antibodies also detected the p53 induction by 5 lM BP clearly. To determine whether the use of whole cell lysates contributed to multiple bands, we compared the immunoblots made from crude lysates against samples where nuclear and cytoplasmic fractions of the mouse and human cells were separated. There was no difference between the results obtained from whole cell and nuclear fractions in immunoblots (data not shown). Monoclonal antibodies PAb 421 or PAb 240 did not produce a visible band at 53 kDa in MCF-7 extracts. Instead, PAb 240 (SCB) revealed one band at 63 kDa, while PAb 240 (ORP) showed a small band at 63 kDa and a strong band at 73 kDa. With BP-treated samples there were also two weak bands between the region from 97 to 220 kDa when PAb 240 (ORP) was used. Computer comparison of PAb 240 with equivalent epitopes in p53 homologues (Table 2) showed over 75% similarity (SAHP53) only with p53 making it unlikely that the homologues interfere with the immunoblotting for p53. With PAb 421 there were three bands at about 35, 43, and 63 kDa and also some background, but no clear bands in the range from 46 to 66 kDa and over
Discussion Despite the wide use of immunological methods to study the p53 protein, papers pursuing the qualitative comparison of various anti-p53 antibodies in immunoblotting are scarce [12–14,22]. Immunoblotting (Western blotting) has been suggested as the ‘‘gold standard’’ for p53 protein expression [11], but there is a lot of confusion in the immunoblotting literature with no explanations e.g., extra bands seen in many immunoblots. Bonsing et al. [12] compared in immunoblotting seven p53 antibodies and several cell lines of which MCF-7 cells and antibodies DO7, 1801, 240, and 421 were also used in this study. Our immunoblotting results in MCF-7 cells using DO7 and PAb 1801 agree well with the results of Bonsing et al., except for the fact that we did not see an 80 kDa band which they saw in the MCF-7 extract with PAb 1801 from Oncogene Science. In accordance with their findings [12], we did not see a visible band at 53 kDa in MCF-7 extracts with monoclonal antibodies PAb 421 or PAb 240. Also in another breast adenocarcinoma cell line (MDA-MB-231), PAb 421 failed to detect p53 in immunoblotting although it worked in immunohistochemistry [22]. Danks et al. [14] could not detect a 53 kDa signal on immunoblots, either, when using PAb 421 against human derived cell lines Rh28 and Dayo. Although, according to the supplier, PAb 421 should be specific for both human and murine p53, it seems to react properly only with mouse p53. The lack of immunoreactivity of PAb 240 with p53 in MCF-7 cells in immunohistochemistry has been reported by Bartek et al. [23]. In our previous studies, we have shown by sequencing exons 5–9, that p53 protein is of wild type in the MCF-7
Table 2 Theoretical probability based on the similarity of amino acids at the site of epitope to detect p53 and related proteins by monoclonal antibodies Gene
Human p53 Mouse p53 Mouse p63 TA g Mouse p63 TA a Mouse p63 DN g Mouse p63 DN a Mouse p73 fragment Human p63 Human p73
Anti-p53 antibody and amino acids of the recognized epitope DO-7 SDLWKL
Pab 421 KKGQSTSRHKK
Pab 240 RHSVV
Pab 1801 SPDDIEQWFT
SIM-score
SAHP53 (%)
SIM-score
SAHP53 (%)
SIM-score
SAHP53 (%)
SIM-score
SAHP53 (%)
34 27 16 18 13 18 15 14 15
100 79 47 53 38 53 44 41 44
57 57 16 21 16 21 17 20 19
100 100 28 37 28 37 30 35 33
25 25 17 17 17 17 17 17 17
100 100 68 68 68 68 68 68 68
59 30 23 23 23 23 15 16 17
100 53 39 39 39 39 25 27 29
(6) (5) (2) (2) (3) (2) (1) (2) (2)
SAHP53 is an Sim-score against human P53.
(11) (11) (4) (4) (4) (4) (3) (3) (3)
(5) (5) (3) (2) (3) (2) (4) (3) (2)
(10) (4) (4) (4) (4) (4) (3) (2) (2)
M. Turpeinen et al. / Biochemical and Biophysical Research Communications 293 (2002) 850–856
cell line we use [18]. Since the epitope of PAb 421 is at the extreme carboxy-terminal part of p53 (Fig. 1), a possible mutation at the epitope region has not been excluded by these studies, because we have sequenced only the central region of p53 in MCF-7 cells. In this study, PAb 240 and PAb 421 antibodies detected a protein in the cells from mouse skin that was slightly larger in size than the band detected by the CM5 antibody. Theoretically, such a shift towards a higher molecular weight can be caused by extensive posttranslational modifications: p53 protein can be phosphorylated [24,25], acetylated [26], and glycosylated [20]. The C-terminal region of human p53 protein is frequently modified post-translationally, possibly masking the PAb 421 epitope. Takenaka et al. [25] have shown that activation of p53 by phosphorylation of serine 378 interferes with the binding of PAb 421 to p53. However, with a high antibody concentration the recognition is retained regardless of phosphorylation [27]. Because we also used a high concentration (low dilution, 1:100) of the PAb 421 in MCF-7 samples, it is unlikely that phosphorylation was the cause of total undetectability of p53 protein by this antibody. In MCF-7 experiments the digestion of putative sugar residues by hexosaminidase treatment of the blots [20,21] did not make the 53 kDa band visible. This rules out the glycosylation of the PAb 421 epitope as an inhibiting factor in the detection. A possible reason for the extra bands in immunoblots is the cross-reactivity with the recently described structural homologues of p53 [28,29]. The p63 and p73 proteins share a close structural homology with p53, especially at the conserved DNA-binding domain and 11 take part in the regulation of normal development and apoptosis. (For recent reviews see [30,31].) Both p63 and p73 have several alternative mRNA isoforms coding proteins that vary at the terminal regions. There are at least four C-terminal variants of p73: a, b, c, and d, while three p63 variants exist differing at the C-terminal ða; b; cÞ, and two at the N-terminal domain (p63TA and p63DN) [32,33]. Using available amino acid sequence information and a computer program we compared the epitopes of the used monoclonal antibodies with the equivalent amino acid sequences of these p53 homologues. Perfect or a close match was found only with the epitopes of p53 making it unlikely that cross-reactivity with the known p53 homologues was the reason for extra bands in immunoblotting. In conclusion, results in p53 immunoblotting vary even when using the same antibody from different commercial companies. Regardless of the recommendations by the manufacturer, careful adjustment of antibody dilutions is needed for each application. A theoretical comparison of an equivalent epitopes of p53 homologues with the known epitopes of p53 antibodies indicates that the extra bands seen with most
855
antibodies are not due to cross-reactivity with the p53 homologues.
Acknowledgments We thank senior laboratory technicians Kirsi Salo and Tuulikki K€arn€a and senior animal technicians Ulla Hirvonen and Tuula Inkala for excellent technical assistance. Prof. David Lane (University of Dundee, Dundee, UK) is gratefully acknowledged for the generous gift of anti-p53 antibodies CM1 and CM5. The work was financially supported by grants from the Cancer Societies of Finland and the Cancer Society of Northern Finland to K.V.
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[29]
[30]
[31] [32] [33]
[34]
[35]
[36]
[37]
[38]
[39]
F. McKeon, D. Caput, Monoallelically expressed gene related to p53 at 1p36 a region frequently deleted in neuroblastoma and other human cancers, Cell 90 (1997) 809–819. A. Yang, M. Kaghad, Y. Wang, E. Gillet, M.D. Fleming, V. Dotsch, N.C. Andrews, D. Caput, F. McKeon, p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing and dominant negative activities, Mol. Cell 2 (1998) 305–316. M. Levrero, V. De Laurenzi, A. Costanzo, S. Sabatini, J. Gong, J.Y.J. Wang, G. Melino, The p/53/p63/p73 family of transcription factors: overlapping and distinct functions, J. Cell Sci. 113 (2000) 1661–1670. M.S. Irwin, W.G. Kaelin, p53 family update: p73 and p63 develop their own identities, Cell Growth Differ. 12 (2001) 337–349. W.G. Kaelin Jr., The p53 gene family, Oncogene 18 (1999) 7701– 7705. P.A. Hall, S.J. Campbell, M. O’Neill, D.J. Royston, K. Nylander, F.A. Carey, N.M. Kernohan, Expression of the p53 homologue p63a and DNp63a in normal and neoplastic cells, Carcinogenesis 21 (2000) 153–160. B. Vojtesek, J. Bartek, C.A. Midgley, D.P. Lane, An immunochemical analysis of the human nuclear phosphoprotein p53. New monoclonal antibodies and epitope mapping using recombinant p53, J. Immunol. Meth. 151 (1992) 237–244. L. Banks, G. Matlashewski, L. Crawford, Isolation of humanp53-specific monoclonal antibodies and their use in studies of human p53 expression, Eur. J. Biochem. 159 (1986) 529–534. H. Wang, J.Y. Wang, L.R. Johnson, R.E. Scott, Selective induction of c-jun and jun-B but not c-fos or c-myc during mitogenesis in SV40-transformed cells at the predifferentiation growth arrest state, Cell Growth Differ. 2 (1991) 645–652. J.V. Gannon, R. Greaves, R. Iggo, D.P. Lane, Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form, EMBO J. 9 (1990) 1595–1602. C.A. Midgley, C.J. Fisher, J. Bartek, B. Vojtesek, D. Lane, D.M. Barnes, Analysis of p53 expression in human tumours: an antibody raised against human p53 expressed Escherichia coli, J. Cell Sci. 101 (1992) 183–189. C.A. Midgley, B. Owens, C.V. Briscoe, D. Brynmor Thomas, D.P. Lane, P.A. Hall, Coupling between gamma irradiation p53 induction and the apoptotic response depends upon cell type in vivo, J. Cell Sci. 108 (1995) 1843–1848.
Comparison of anti-p53 antibodies in immunoblotting Miia Turpeinen, Raisa Serpi, Mika Rahkolin, and Kirsi V€ ah€ akangas* Department of Pharmacology and Toxicology, P.O. Box 5000, FIN-90014 University of Oulu, Oulu, Finland Received 25 March 2002
Abstract Some of the most important tools to study p53 protein are various anti-p53 antibodies and immunological methods based on antibody–antigen reactions. Critical comments on the specificity and sensitivity of anti-p53 antibodies have been published. Four monoclonal and two polyclonal anti-p53 antibodies, four of them from two different sources, were compared for their ability to detect in immunoblotting the benzo(a)pyrene-induced p53 from C57BL/6 mouse skin and MCF-7 human breast carcinoma cells. Multiple extra bands were seen with most antibodies. A theoretical comparison of the equivalent epitopes of p53 homologues with the known epitopes of p53 antibodies indicated that the extra bands seen with most antibodies are not due to cross-reactivity with these homologues. A careful adjustment of antibody dilutions is needed for each application utilizing commercial p53 antibodies, regardless of the recommendations of the supplier. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Immunoblotting; Anti-p53 antibodies; MCF-7 cell line; Mouse skin; Epitopes; p53 Homologues
p53 is one of the most studied proteins because of its crucial role in the cell cycle and the fact that over 50% of all cancers contain a mutation in TP53 gene [1–3]. As a transcription factor p53 induces cell cycle arrest, DNAdamage repair, and apoptosis by increasing the expression of a number of other genes such as p21/WAFl, BAX, GADD45, TFIID, TFIIH, MDM2, and ARF [4,5]. Human p53 protein consists of 393 and mouse p53 protein of 390 amino acid residues. p53 protein can be divided into five different functional and structural domains [4]. The N-terminal end of p53 acts as a transactivation area (amino acids 1–42), which binds at the promoter regions the regulatory sequences of its target genes. Carboxy-terminal domain of p53 (amino acid residues 360–393) regulates the activity of p53 protein and binds DNA non-specifically. The highly conserved central core region between amino acids 100 and 300 binds DNA in a sequence-specific manner [6,7]. The majority of known p53 mutations are located within this
*
Corresponding author. Permanent address: Department of Pharmacology and Toxicology, University of Kuopio, P.O. Box 1627, FIN70211 Kuopio, Finland. Fax: +358-17-162-424/+358-8-537-5247. E-mail addresses: kirsi.vahakangas@oulu.fi, kirsi.vahakangas@ uku.fi (K. V€ ah€ akangas).
core region of the protein [2,3]. Most of the antibody epitopes as well as phosphorylation and acetylation sites are found at the ends of the p53 protein [8,9]. Posttranslational modifications have importance in stabilization and activation of a p53 protein [10]. Immunoblotting has been suggested ‘‘as the gold standard for demonstrating p53 expression’’ [11]. However, many anti-p53 antibodies used in immunoblotting have not performed according to the expectations. Critical comments on p53 antibodies and unidentified extra bands have been reported [12–15]. For a comparison of the results between laboratories there is clearly a need for more intra- and inter-laboratory validation of the immunomethods. We have compared in immunoblotting six different anti-p53 antibodies, suitable for this purpose according to the supplier. The detectability of p53 protein obtained from MCF-7 cells and mouse skin cells treated by benzo(a)pyrene was studied. We have shown before that p53 protein is inducible by benzo(a)pyrene in these models [16–19]. Materials and methods Antibodies. Anti-p53 antibodies PAb 421, CM1, DO7, PAb 1801, and PAb 240 were evaluated for their ability to detect p53 from MCF-7 human breast cancer cells. PAb 240, PAb 421, and CM5
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 2 9 8 - X
M. Turpeinen et al. / Biochemical and Biophysical Research Communications 293 (2002) 850–856 antibodies were used for p53 from C57BL/6 mouse skin cells (Fig. 1, Table 1). All of the commercial antibodies are recommended, among other applications, also for immunoblotting. CM5 and one of the two CM1 antibodies used were original antibodies from Prof. David Lane. Other antibodies were from commercial suppliers. All antibodies were titrated to reach the optimal background and visibility of the p53 band. Secondary antibody used against mouse monoclonal antibodies DO7, PAb 1801, PAb 240, and PAb 421 was a polyclonal sheep anti-mouse IgG conjugated with horseradish per-
851
oxidase (Batch 144832, Amersham Life Science, Buckinghamshire, UK). The secondary antibody against antibodies CM1 and CM5 was a polyclonal goat anti-rabbit antibody conjugated with horseradish peroxidase (Lot# B16624, Oncogene Research Products, Cambridge, MA). Mice. Male C57BL/6 mice (12–15 weeks of age) were maintained in plastic cages at a constant temperature of 25 °C with a 12 h lightdark-cycle and had free access to food (Standard rodent pellets, Special Diets Services, Essex, England) and water. The backs of the mice were
Fig. 1. Epitopes for anti-p53 antibodies used in this study. Functional domains of p53 protein are indicated by boxes I transactivation domain, II proline-rich region, III specific DNA-binding domain, IV oligomerization domain, V basic C-terminal domain. Amino acid numbers for both functional areas and antibody epitopes are indicated.
Table 1 Antibodies used in this study Primary antibody
Manufacturer
Type of antibody
Specificity
Used working dilution
Recommended working dilution
Reference
MCF-7 DO7 DO7 PAb 1801 PAb 1801 PAb 421
NCL ST ORP SCB ORP
Mouse Mouse Mouse Mouse Mouse
1:2000 1:1000 1:1000 1:500 1:100
1:1000–1:5000 1:1000 2:5 lg=ml (1:400) – 10 lg=ml (1:100)
[34] [34] [35] [34] [36]
PAb 240
SCB
Mouse monoclonal
1:200
–
[34]
PAb 240 CM1 CM1
ORP NCL
Mouse monoclonal Rabbit polyclonal Rabbit polyclonal
Human wt/mut Human wt/mut Human wt/mut Human Human, rat, mouse, monkey rabbit Human, rat, mouse, avian, bovine Human, mouse Human wt/mut Human
1:200 1:16,000 1:32,000
5 lg=ml (1:200) 1:500–1:1000 –
[37] [38] [38]
1:2000
10 lg=ml (1:100)
[36]
1:4000 1:4000 1:2000
– 5 lg=ml (1:200) –
[34] [37] [39]
Mouse skin PAb 421 PAb 240 PAb 240 CMS
a
monoclonal monoclonal monoclonal monoclonal monoclonal
ORP
Mouse monoclonal
SCB ORP
Mouse monoclonal Mouse monoclonal Rabbit polyclonal
a
Human, rat, mouse, monkey rabbit Human, mouse Human, mouse Mouse
NCL, Novocastra Laboratories (UK); ST, Serotec (UK); ORP, Oncogene Research Products (MA, USA); SCB, Santa Cruz Biotechnology (CA, USA). a Gift from Prof. David Lane (Dundee, UK).
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shaved three days before the treatments and only mice with fur in a non-growing phase were selected. Five hundred micrograms of benzo(a)pyrene (BP) (Sigma, St. Louis, MO) in 100 ll of acetone was applied on the shaved backs of the mice. Control animals were treated with 100 ll of acetone. Skin samples were collected 24 h after the treatment when the p53 response was strongest [16,17]. For immunoblotting analysis the treated mouse skin was removed and subcutaneous fat was removed on ice. The mouse skin samples were homogenized on ice for 10 min in 1.5 ml nuclear lysis buffer (20% glycerol, 20 mM HEPES, 500 mM NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 1 mM DTT, 0.1% NP40, 100 lg=ml PMSF, 1 lg=ml aprotinin, 1 lg=ml pepstatin A, and 1 lg=ml antipain). The samples were incubated for 20 min on ice and then centrifugated for 15 min at 12,000 rpm at +4 °C. The supernatant (whole cell extract) was collected and the samples were maintained at )70 °C until needed. MCF-7 cells. MCF-7 cells, which express wild type p53 protein [18], were cultured at 37 °C in an atmosphere of 95% humified air and 5% CO2 . The used RPMI 1640 medium contained non-essential amino acids, 10% fetal bovine serum, 1 mM streptomycin, 1 mM penicillin, 2 mM glutamine (all from Gibco, Paisley, UK), 1 lg=ml estradiol (Sigma, St. Louis, MO), and 1 lg=ml insulin (Novo Nordisk, Bagsværd, Danmark). To increase the amount of p53 protein subconfluent cell cultures were treated with either 1 lM or 5 lM benzo(a)pyrene (BP) (Sigma, St. Louis, MO). BP was diluted in acetone and 50 ll of the dilution was added to the medium for 48 h [18]. The control cells were treated with acetone. For immunoblotting, MCF-7 cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in 150 ll nuclear lysis buffer. The samples were incubated for 20 min on ice and then centrifuged for 15 min at 12,000 rpm at +4 °C. The supernatant (whole cell extract) was collected and the samples were maintained at )70 °C until needed. Immunoblotting (Western blotting). Thirty micrograms of protein (50 lg of MCF-7-samples for PAb 240 and PAb 421) was heated for 4 min at 96 °C in Laemmli buffer and separated electrophoretically at 200 V (Bio-Rad Power Pac 200, Bio Rad, Hercules, CA) in a 10% polyacrylamide gel containing SDS. Polyvinylidene difluoride (PVDF) filter (Immobilon P, 0.45 lm pore size, Millipore, Bedford, MA) was treated according to the instructions of the manufacturer and the proteins were transferred to a filter by electroblotting for 1 h at 100 V. To avoid unspecific binding, the filters were blocked overnight at +4 °C in Tris-buffered saline (TBS) containing 5% non-fat cow milk powder. The filters were incubated in a primary antibody at room temperature for 1 h, washed five times with TBS containing 0.05% Tween 20, incubated for 1 h in a secondary antibody at room temperature and washed again thoroughly with TBS–Tween. Enhanced chemiluminescence system (ECL+, Amersham Life Science, Buckinghamshire, UK) was used according to the instructions of the manufacturer. The size of a band was defined by Rainbow Coloured Protein Molecular Weight Marker (Amersham Life Science, Buckinghamshire, UK) analysed in parallel with the samples. Immunoblotting was repeated from two to nine times depending on the antibody and only reproducible results are reported. For comparisons, the same three samples of MCF-7 and the same two samples from mouse skin were used throughout the experiments. Hexosaminidase treatment to digest the possible sugar residues in the blots was done after the blocking of unspecific binding in milk according to Shaw et al. [20] and Jackson and Tjian [21]. Briefly, the filters were rinsed quickly with 50 mM sodium citrate, pH 5.0 (Sigma, St. Louis, MO) and then incubated at 37 °C for 3, 6, 12, or 24 h with 2.5 units of b-N-acetylglucosaminidase (Sigma, St. Louis, MO) in 5 ml sodium citrate. The blots were then extensively washed with TBS– Tween, re-equilibrated in 5% TBS–milk and then blotted with the primary antibody as above. Computer analysis of the protein sequences. Protein sequences were obtained by using SWISS-PROT/TrEMBL—database (http:// www.expasy.ch/cgi-bin/sprot-search-de). All sequences were compared against the theoretical epitope area of each antibody by using SIM-
Alignment analysis tool for protein sequences (http://www.expasy.ch/ tools/sim-prot.html). The sequences used were for mouse O70366 (p53), O88897 (p63 TA c), O88898 (p63 TA a), O88899 (p63 DN c), O89097 (p63 DN a), and Q9WUJO (p73 fragment) and for human P04637 (p53), Q07065 (p63), and O15350 (p73). Program was used by default parameters (Comparison matrix BLOSUM62, gap open penalty 12, gap extension penalty 4). Comparison of the sequences is expressed as an SIM-score, which is SIM-program’s best score for alignment of two sequences. For instance, the score for the amino acid sequence of DO7 epitope and human p53 is 34 which is reached when all six amino acids of the epitope are in correct order and detectable in the protein. To compare the theoretical possibilities of different proteins to be detectable by a certain antibody, a percentage of the SIMscore from a perfect match (human p53) was calculated (SAHP53).
Results The dilutions of the antibodies recommended by the suppliers were not the best in all cases for the ECL+ based detection of p53 (Table 1). In mouse skin cells, all the antibodies used (PAb 240 from two commercial sources, PAb 421 and CM5) detected p53 protein and its increase by BP treatment (Fig. 2). However, the intensity of the bands and the strength of the induction differed depending on the antibody. CM5, which did not detect any band at 53 kDa in the untreated skin, and PAb 240 from SCB showed the clearest indication of p53 response, while with PAb 421 the induction was barely detectable. PAb 421 and both PAb 240 antibodies detected a protein slightly larger in size than the band detected by CM5.
Fig. 2. Immunoblots from untreated (lane 1) and benzo(a)pyrenetreated (lane 2: 500 lg for 24 h) skin from C57BL/6 mice. (a) CM5; (b) PAb 421 (ORP); (c) PAb 240 (ORP) and (d) PAb 240 (SCB). The used working dilution for each antibody is presented in Table 1. Thirty micrograms of protein was used for all antibodies. p53-Band is indicated with an arrow.
M. Turpeinen et al. / Biochemical and Biophysical Research Communications 293 (2002) 850–856
In skin cells from in vivo-treated mice all four antibodies showed a strong band of approximately 66 kDa. This band was similar in intensity to BP-treated and control samples. CM5 also showed a smaller band of 46 kDa and PAb 240, and PAb 421 a weak band of
853
60 kDa. There was no difference in the intensities of these bands between treated and control samples. In MCF-7 samples the p53 band was detectable when antibodies CM1, PAb 1801, and DO7 were used (Fig. 3). Multiple bands with the polyclonal antibody CM1 (from
Fig. 3. Immunoblots from untreated (lane 1) and benzo(a)pyrene-treated (lane 2: 1 lM for 48 h; lane 3: 5 lM for 48 h) MCF-7 cells: (a) DO7 (NCL); (b) DO7 (ST); (c) PAb 1801 (ORP); (d) PAb 1801 (SCB); (e) CM1 (NCL); (f) CM1 (a gift from Prof. David Lane, Dundee, UK); (g) PAb 240 (ORP); (h) PAb 240 (SCB); (i) PAb 421 (ORP). The used working dilution for each antibody is presented in Table 1. Thirty micrograms of protein was used for a–f and 50 lg for g–i. The site of 53 kDa band is indicated with an arrow.
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97 kDa. Computer analysis of PAb 421 showed that the site of the epitope in p53-related proteins was less than 40% similar with the epitope of p53. Thus the probability that PAb 421 detects other than p53 protein is small.
NCL and Prof. David Lane) are easily explained because each polyclonal antibody is a serum of the immunized animal and thus contains antibodies against many epitopes. With both of the CM1 antibodies the main bands were at about 35, 48, 53, 63, and 66 kDa. Some weak bands between these regions were also detectable through the whole area. The benzo(a)pyrene induction was also seen with CM1 after 5 lM BP treatment, although not very strongly. Both DO7 antibodies (ST, NCL) gave one sharp band in MCF-7 cells at 53 kDa while PAb 1801 antibodies showed bands at 53 and 46 kDa and a weak band at about 63 kDa. PAb 1801 (ORP) detected also a band at 66 kDa. The dosedependent p53 response was most obvious with the DO7 antibody. With the DO7 from NCL the induction of p53 protein was seen already with 1 lM BP, while the DO7 from ST showed an increase only with 5 lM BP. Both PAb 1801 antibodies also detected the p53 induction by 5 lM BP clearly. To determine whether the use of whole cell lysates contributed to multiple bands, we compared the immunoblots made from crude lysates against samples where nuclear and cytoplasmic fractions of the mouse and human cells were separated. There was no difference between the results obtained from whole cell and nuclear fractions in immunoblots (data not shown). Monoclonal antibodies PAb 421 or PAb 240 did not produce a visible band at 53 kDa in MCF-7 extracts. Instead, PAb 240 (SCB) revealed one band at 63 kDa, while PAb 240 (ORP) showed a small band at 63 kDa and a strong band at 73 kDa. With BP-treated samples there were also two weak bands between the region from 97 to 220 kDa when PAb 240 (ORP) was used. Computer comparison of PAb 240 with equivalent epitopes in p53 homologues (Table 2) showed over 75% similarity (SAHP53) only with p53 making it unlikely that the homologues interfere with the immunoblotting for p53. With PAb 421 there were three bands at about 35, 43, and 63 kDa and also some background, but no clear bands in the range from 46 to 66 kDa and over
Discussion Despite the wide use of immunological methods to study the p53 protein, papers pursuing the qualitative comparison of various anti-p53 antibodies in immunoblotting are scarce [12–14,22]. Immunoblotting (Western blotting) has been suggested as the ‘‘gold standard’’ for p53 protein expression [11], but there is a lot of confusion in the immunoblotting literature with no explanations e.g., extra bands seen in many immunoblots. Bonsing et al. [12] compared in immunoblotting seven p53 antibodies and several cell lines of which MCF-7 cells and antibodies DO7, 1801, 240, and 421 were also used in this study. Our immunoblotting results in MCF-7 cells using DO7 and PAb 1801 agree well with the results of Bonsing et al., except for the fact that we did not see an 80 kDa band which they saw in the MCF-7 extract with PAb 1801 from Oncogene Science. In accordance with their findings [12], we did not see a visible band at 53 kDa in MCF-7 extracts with monoclonal antibodies PAb 421 or PAb 240. Also in another breast adenocarcinoma cell line (MDA-MB-231), PAb 421 failed to detect p53 in immunoblotting although it worked in immunohistochemistry [22]. Danks et al. [14] could not detect a 53 kDa signal on immunoblots, either, when using PAb 421 against human derived cell lines Rh28 and Dayo. Although, according to the supplier, PAb 421 should be specific for both human and murine p53, it seems to react properly only with mouse p53. The lack of immunoreactivity of PAb 240 with p53 in MCF-7 cells in immunohistochemistry has been reported by Bartek et al. [23]. In our previous studies, we have shown by sequencing exons 5–9, that p53 protein is of wild type in the MCF-7
Table 2 Theoretical probability based on the similarity of amino acids at the site of epitope to detect p53 and related proteins by monoclonal antibodies Gene
Human p53 Mouse p53 Mouse p63 TA g Mouse p63 TA a Mouse p63 DN g Mouse p63 DN a Mouse p73 fragment Human p63 Human p73
Anti-p53 antibody and amino acids of the recognized epitope DO-7 SDLWKL
Pab 421 KKGQSTSRHKK
Pab 240 RHSVV
Pab 1801 SPDDIEQWFT
SIM-score
SAHP53 (%)
SIM-score
SAHP53 (%)
SIM-score
SAHP53 (%)
SIM-score
SAHP53 (%)
34 27 16 18 13 18 15 14 15
100 79 47 53 38 53 44 41 44
57 57 16 21 16 21 17 20 19
100 100 28 37 28 37 30 35 33
25 25 17 17 17 17 17 17 17
100 100 68 68 68 68 68 68 68
59 30 23 23 23 23 15 16 17
100 53 39 39 39 39 25 27 29
(6) (5) (2) (2) (3) (2) (1) (2) (2)
SAHP53 is an Sim-score against human P53.
(11) (11) (4) (4) (4) (4) (3) (3) (3)
(5) (5) (3) (2) (3) (2) (4) (3) (2)
(10) (4) (4) (4) (4) (4) (3) (2) (2)
M. Turpeinen et al. / Biochemical and Biophysical Research Communications 293 (2002) 850–856
cell line we use [18]. Since the epitope of PAb 421 is at the extreme carboxy-terminal part of p53 (Fig. 1), a possible mutation at the epitope region has not been excluded by these studies, because we have sequenced only the central region of p53 in MCF-7 cells. In this study, PAb 240 and PAb 421 antibodies detected a protein in the cells from mouse skin that was slightly larger in size than the band detected by the CM5 antibody. Theoretically, such a shift towards a higher molecular weight can be caused by extensive posttranslational modifications: p53 protein can be phosphorylated [24,25], acetylated [26], and glycosylated [20]. The C-terminal region of human p53 protein is frequently modified post-translationally, possibly masking the PAb 421 epitope. Takenaka et al. [25] have shown that activation of p53 by phosphorylation of serine 378 interferes with the binding of PAb 421 to p53. However, with a high antibody concentration the recognition is retained regardless of phosphorylation [27]. Because we also used a high concentration (low dilution, 1:100) of the PAb 421 in MCF-7 samples, it is unlikely that phosphorylation was the cause of total undetectability of p53 protein by this antibody. In MCF-7 experiments the digestion of putative sugar residues by hexosaminidase treatment of the blots [20,21] did not make the 53 kDa band visible. This rules out the glycosylation of the PAb 421 epitope as an inhibiting factor in the detection. A possible reason for the extra bands in immunoblots is the cross-reactivity with the recently described structural homologues of p53 [28,29]. The p63 and p73 proteins share a close structural homology with p53, especially at the conserved DNA-binding domain and 11 take part in the regulation of normal development and apoptosis. (For recent reviews see [30,31].) Both p63 and p73 have several alternative mRNA isoforms coding proteins that vary at the terminal regions. There are at least four C-terminal variants of p73: a, b, c, and d, while three p63 variants exist differing at the C-terminal ða; b; cÞ, and two at the N-terminal domain (p63TA and p63DN) [32,33]. Using available amino acid sequence information and a computer program we compared the epitopes of the used monoclonal antibodies with the equivalent amino acid sequences of these p53 homologues. Perfect or a close match was found only with the epitopes of p53 making it unlikely that cross-reactivity with the known p53 homologues was the reason for extra bands in immunoblotting. In conclusion, results in p53 immunoblotting vary even when using the same antibody from different commercial companies. Regardless of the recommendations by the manufacturer, careful adjustment of antibody dilutions is needed for each application. A theoretical comparison of an equivalent epitopes of p53 homologues with the known epitopes of p53 antibodies indicates that the extra bands seen with most
855
antibodies are not due to cross-reactivity with the p53 homologues.
Acknowledgments We thank senior laboratory technicians Kirsi Salo and Tuulikki K€arn€a and senior animal technicians Ulla Hirvonen and Tuula Inkala for excellent technical assistance. Prof. David Lane (University of Dundee, Dundee, UK) is gratefully acknowledged for the generous gift of anti-p53 antibodies CM1 and CM5. The work was financially supported by grants from the Cancer Societies of Finland and the Cancer Society of Northern Finland to K.V.
References [1] M. Hollstein, D. Sidransky, B. Vogelstein, C.C. Harris, p53 mutations in human cancers, Science 253 (1991) 49–53. [2] A.J. Levine, J. Momand, C.A. Finlay, The p53 tumour suppressor gene, Nature 351 (1991) 453–456. [3] M.S. Greenblatt, W.B. Bennett, M. Hollstein, C.C. Harris, Mutations in the p53 tumour suppressor gene: clues to cancer etiology and molecular pathogenesis, Cancer Res. 54 (1994) 4855– 4878. [4] C. Prives, P.A. Hall, The p53 pathway, J. Pathol. 187 (1999) 112– 126. [5] E.E. Balint, K.H. Vousden, Activation and activities of the p53 tumour suppressor protein, Br. J. Cancer 12 (2001) 1813–1823. [6] L.J. Ko, C. Prives, p53: puzzle and paradigm, Genes Dev. 10 (1996) 1054–1072. [7] K.G. Wiman, p53: emergency brake and target for cancer therapy, Exp. Cell Res. 237 (1997) 14–18. [8] Y. Legros, C. Lafon, T. Soussi, Linear antigenic sites defined by the B-cell response to human p53 are localized predominantly in the amino and carboxy-termini of the protein, Oncogene 9 (1994) 2071–2076. [9] D.P. Lane, C.W. Stephen, C.A. Midgley, A. Sparks, T.R. Hupp, D.A. Daniels, R. Greaves, A. Reid, B. Vojtesek, S.M. Picksley, Epitope analysis of the murine p53 tumour suppressor protein, Oncogene 12 (1996) 2461–2466. [10] D.B. Woods, K.H. Vousden, Regulation of p53 function, Exp. Cell Res. 264 (2001) 56–66. [11] A. Nickels, H. Selter, M. Pfreundschuh, M. Montenarh, B. Koch, Detection of p53 in inflammatory tissue and lymphocytes using immunohistology and flow cytometry: a critical comment, J. Clin. Pathol. 50 (1997a) 654–660. [12] B.A. Bonsing, W.E. Corver, M.C.B. Gorsira, M. van Vliet, P.S. Oud, C.J. Cornelisse, G.J. Fleuren, Specificity of seven monoclonal antibodies against p53 evaluated with western blotting, immunohistochemistry, confocal laser scanning, microscopy and flow cytometry, Cytometry 28 (1997) 11–24. [13] A. Nickels, M. Bureik, M. Montenarh, M. Pfreundschuh, B. Koch, p53 antibodies: call for quality. Letter to the editor, Int. J. Cancer 73 (1997b) 613–614. [14] M.K. Danks, D.O. Whipple, C.R. McPake, D. Lu, L.C. Harris, Differences in epitope accessibility of p53 monoclonal antibodies suggest at least three conformations or states of protein binding of p53 protein in human tumor cell lines, Cell Death Differ. 5 (1998) 678–686. [15] A. Harlozinska, J. Bar, M. Montenarh, Analysis of the immunoreactivity of three anti-p53 antibodies and estimation of the relations between p53 status and MDM2 protein expression in ovarian carcinomas, Anticancer Res. 20 (2000) 1049–1056.
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