- Email: [email protected]
Documentation of Negatively Stained Polyacrylamide Gels
237
NOTES & TIPS
FIG. 2. The integrity of proteins on the blot are preserved better with the non-block technique. Lysates of Jurkat T cells prepared in 1% NP-40 lysis buffer (50 g per lane) were loaded onto a 10% SDS–polyacrylamide gel and separated under reducing conditions. After the transfer of the separated proteins onto a PVDF membrane, the blots were probed with a mix of anti-ZAP70 (1:500 dilution) and anti-p38 MAPK (1:1000 dilution) antibodies in TBS-T. The membranes were then stripped of the probing antibodies. To check for the completeness of the stripping protocol, the membranes were reprobed with anti-rabbit antibodies and visualized by ECL (middle). After four more strippings, the membranes were each reprobed with the same mix of anti-ZAP70 and anti-p38 MAPK antibodies as previously. For both the conventional and the non-block techniques the exposure times for the detection of the ECL signal were the same. Identical probing and stripping conditions were used for both methods.
stroyed the immunorecognition epitopes of the blotted proteins (unpublished observations). We conclude that with the stripping protocol presented here the non-block technique of Western probing can be used for multiple sequential probing of the same blot with little or no loss of signal. This may be critical in cases where multiple probing of a valuable blot is necessary and maximal detection sensitivity is required. Acknowledgments. This work was supported by Grant RO1 AI26644 from the National Institutes of Health and by the Rosalind Russell Arthritis Center.
Documentation of Negatively Stained Polyacrylamide Gels 1 Terry M. Bricker,* Kari B. Green-Church,† Patrick A. Limbaugh,† and Laurie K. Frankel* *Department of Biological Sciences, Division of Biochemistry and Molecular Biology, and †Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803 Received November 16, 1999
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Mansfield, M. A. (1995) Anal. Biochem. 229, 140 –143. Stott, D. I. (1989) J. Immunol. Methods 119, 153–187. Laemmli, U. K. (1970) Nature 227, 680 – 685. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. Sadra, A., Cinek, T., Arellano, J. L., Shi, J., Truitt, K. E., and Imboden, J. B. (1999) J. Immunol. 162, 1966 –1973. Schneider, H., Cai, Y. C., Prasad, K. V., Shoelson, S. E., and Rudd, C. E. (1995) Eur. J. Immunol. 25, 1044 –1050. Sutherland, C. L., Krebs, D. L., and Gold, M. R. (1999) J. Immunol. 162, 4720 – 4730. Suck, R. W. L., and Krupinska, K. (1996) BioTechniques 21, 418 – 422.
Heavy metal negative staining (1, 2) is becoming increasingly popular for the detection of proteins separated by SDS–polyacrylamide gel electrophoresis. Advantages of the method include ease of use (rapid staining with destaining not being required), high sensitivity (intermediate to that exhibited by Coomassie blue and silver staining), reversibility (3), compatibility with Western blotting and subsequent immunodetection (4, 5), and 1 Funding was provided by the generous support of the National Science Foundation and the Department of Energy to T.M.B. and L.K.F. and the National Institutes of Health to P.A.L.
Analytical Biochemistry 278, 237–239 (2000) doi:10.1006/abio.1999.4446 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
238
NOTES & TIPS
compatibility with mass spectrometric procedures (6). Generally, SDS–polyacrylamide gels are soaked for a brief time in the chloride or sulfate salts of copper or zinc. The heavy metal salt forms an insoluble precipitate with the SDS. In some protocols the gels are pretreated with 200 mM imidazole (2), which provides a more homogeneous background. In all of these methods, the protein bands appear as clear zones embedded in a milky white background. While this method is becoming increasingly popular, the photographic documentation of gels stained in this manner is problematic. The gels inherently exhibit low contrast and are usually photographed with indirect lighting against a black background. It is often difficult to achieve even lighting across the entire gel, which leads to an apparent unevenness in the light-scattering of the background and poor image quality. Transmitted light photography, which is invariably used to document Coomassie blue-stained or silver-stained polyacrylamide gels, is inappropriate for use with negatively stained gels because the background transmits scattered light efficiently, leading to unacceptably low contrast images. In our laboratory, we have found that these gels can be easily documented by the use of a low-cost flatbed digital scanner. These devices utilize reflected rather than transmitted light to acquire an image. The milky white background of the negatively stained gels efficiently back-scatters light while the clear protein bands do not. When scanned with a black background (which is standard with most flatbed scanners), the protein bands appear dark against a white background. To demonstrate the utility of this documentation method, varying concentrations of standard proteins (Sigma Chemical Corp., SDS-6H) were separated on a 10% polyacrylamide gel using the buffer system of Laemmli (7). The proteins were electrophoresed overnight at 0.5 W. After electrophoresis, the gel was rinsed briefly in deionized water, and immersed in 300 mM CuCl 2 with gentle shaking for about 5 min. After full development of the negative image, the gel was rinsed briefly in deionized water and placed directly on the glass bed of a Microtek Scanmaker E6 digital scanner. The wet gel was overlayed with a piece of transparent acetate and the lid of the scanner, which contains the black background, was placed gently over the gel. The gel was scanned at 600 ⫻ 600 dpi resolution and either 256-level gray scale or 24-bit color resolution. Data acquisition times were 90 s for gray scale and 260 s for color. The image was acquired with PhotoImpact, Ver. 3.0 (Ulead Systems, Inc.) software and saved as a tagged-information format (tif) file. Contrast and brightness levels were adjusted to yield an image which closely corresponded to the visual appearance of the negatively stained polyacrylamide gel. For publication, the gel image was printed on an Epson 900
FIG. 1. Molecular weight standards separated by SDS–PAGE and copper-stained. After staining, the gel was scanned as described in the text, imported into Corel Draw 8.0 for labeling, and printed with an Epson 900 ink jet printer. The amount of standard protein in each protein band is shown above, while the molecular masses of the standard proteins are shown to the right.
ink jet printer on Kodak glossy ink jet paper after labeling with Corel Draw 8.0 (Corel Corp.). The results of this method of gel documentation are shown in Fig. 1. The image was captured using 256 gray scale levels and yielded an image file of 7 MB. The protein bands, which are transparent, do not efficiently scatter the light from the bulb of the digital scanner and appear as dark bands while the SDS– copper precipitate does back-scatter the light from the scanner and appears milky white. The image contrast is quite good, with the protein bands clearly visible in the scanned image. The results obtained with 24-bit color were comparable to the gray-scale image, exhibiting subjectively slightly improved resolution (data not shown). This slight image improvement was offset by a much larger image data file (18 MB) and consequently slower image processing times. While the results obtained from this method are clearly suitable for publication, it should be noted that the primary intent of this communication is to provide a simple, quick, and inexpensive method for the day-to-day documentation of negatively stained polyacrylamide gels. For most purposes, Coomassie blue staining or silver staining, followed by classical photography or digitization and image processing, provides superior images for publication. It should be noted that negative staining procedures have also been developed for use in detecting nucleic acids separated in either agarose or polyacrylamide gels (8). Our procedure should also be useful for the documentation of gels stained by these methods.
NOTES & TIPS
REFERENCES 1. Lee, C., Levin, A., and Branton, D. (1987) Copper staining: a five-minute protein stain for sodium dodecyl sulfate–polyacrylamide gels. Anal. Biochem. 1, 308 –312. 2. Fernandez-Patron, C., Castellanos-Serra, L., and Rodriguez, P. (1992) Reverse staining of sodium dodecyl sulfate–polyacrylamide gels by imidazole–zinc salts: Sensitive detection of unmodified proteins. Biotechniques 12, 564 –573. 3. Hardy, E., Santana, H., Hernandez, L., Fernandez-Patron, C., and Castellanos-Serra, L. (1996) Recovery of biologically active proteins detected with imidazole–sodium dodecyl sulfate–zinc (reverse stain) on sodium dodecyl sulfate gels. Anal. Biochem. 240, 150 –152. 4. Tessmer, U., and Dernick, R. (1989) Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate– polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of specific antisera. Electrophoresis 10, 277–279. 5. Wang, D., Dzandu, J. K., Hussain, M., and Johnson, R. M. (1989) Western blots from sodium dodecyl sulfate–polyacrylamide gels stained by metal salts. Anal. Biochem. 180, 311–313. 6. Cohen, S. L., and Chait, B. T. (1997) Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate–polyacrylamide gels. Anal. Biochem. 247, 257–267. 7. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680 – 685. 8. Hardy, E., Pupo, E., Casalvilla, R., Sosa, A. E., Trujillo, L. E., Lopez, E., and Castellanos-Serra, L. (1996) Negative staining with zinc–imidazole of gel electrophoresis-separated nucleic acids. Electrophoresis 17, 1537–1541.
Use of Hydrophobic Interaction Chromatography to Separate Recombinant Antibody Fragments from Associated Bacterial Chaperone Protein GroEL Kevin C. O’Connor,* ,1 Shibnath Ghatak,† and B. David Stollar* ,2 *Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, and †Division of Hematology/Oncology, Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts 02111 Received December 8, 1999
Many systems for expression of recombinant antibodies and antibody fragments have been developed in 1 Current address: Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Institutes of Medicine, Room 780, 77 Avenue Louis Pasteur, Boston, MA 02115. 2 To whom correspondence and reprint requests should be addressed at Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. Fax (617) 6362409. E-mail: [email protected].
Analytical Biochemistry 278, 239 –241 (2000) doi:10.1006/abio.1999.4465 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
239
the past decade. These systems enable rapid investigation into contributions of individual heavy and light chain V regions or particular amino acid residues to antigen recognition and binding. Whole antibody molecules have been expressed in plasmid-transfected mammalian (1) or baculovirus-infected insect cells (2), and Fab or Fv fragments have been expressed in yeast (3) or bacteria (4, 5). Bacterial systems have been especially popular because of their ease of genetic manipulation, efficient transformation, quick growth, and low cost. They have been used for production of Fabs (4), two-chain Fv (6), or scFv 3 (5, 7) domains, including those of both autoantibodies (8, 9) and immunizationinduced antibodies to DNA (10). We constructed plasmids pIg16 and pIg20 for the expression of scFv, VH, or VL domains fused to a single B domain of staphylococcal protein A (SPA) (10 –12). The vectors, derived from pGEMEX-1 (Promega, Madison, WI), use a T7 RNA polymerase promoter to drive transcription. The host cell is the BL21(DE3)–pLysE strain of Escherichia coli, engineered so as to contain the T7 RNA polymerase gene under lac promoter control (13, 14). Upon induction with IPTG, T7 RNA polymerase is produced and transcribes the pIg vector insert from the T7 promoter. A constitutively expressed plasmid pLysE, with a chloramphenicol resistance marker, is also present in these BL21 cells to ensure tight control of polymerase activation; a small amount of polymerase that may be formed before induction is bound and inactivated by the lysozyme produced from pLysE (13). Secretion of the expressed protein coded in the pIg vectors results from the presence of a bacterial alkaline phosphatase (Pho A) leader peptide coded at the 5⬘ end of the construct. In most cases, recombinant antibody– SPA fusion proteins are readily purified from clarified bacterial growth medium with an affinity chromatography column bearing IgG, to which the proteins bind through their SPA domains. The scFv–SPA, VH–SPA, or VL–SPA usually gives a single band on a Coomassie blue-stained SDS–PAGE gel (15). Sometimes, however, a bacterial protein copurifies with the antibody fragment. We have identified the bacterial protein as GroEL and have tested methods for separating the antibody fragment from it. We studied four preparations that contained bacterial protein along with scFv–SPA after they were purified from culture supernatants by capture on IgG– Sepharose (Pharmacia, Piscataway, NJ). They were derived from monoclonal antibodies dC1, dC5 (both anti-poly(dC)), 5E3 (ligand unknown), and SG45 (antiCD45). Migration of each of the scFv domains on SDS– 3 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; IPTG, isopropylthiogalactoside; scFv, single chain Fv domain.
NOTES & TIPS
FIG. 2. The integrity of proteins on the blot are preserved better with the non-block technique. Lysates of Jurkat T cells prepared in 1% NP-40 lysis buffer (50 g per lane) were loaded onto a 10% SDS–polyacrylamide gel and separated under reducing conditions. After the transfer of the separated proteins onto a PVDF membrane, the blots were probed with a mix of anti-ZAP70 (1:500 dilution) and anti-p38 MAPK (1:1000 dilution) antibodies in TBS-T. The membranes were then stripped of the probing antibodies. To check for the completeness of the stripping protocol, the membranes were reprobed with anti-rabbit antibodies and visualized by ECL (middle). After four more strippings, the membranes were each reprobed with the same mix of anti-ZAP70 and anti-p38 MAPK antibodies as previously. For both the conventional and the non-block techniques the exposure times for the detection of the ECL signal were the same. Identical probing and stripping conditions were used for both methods.
stroyed the immunorecognition epitopes of the blotted proteins (unpublished observations). We conclude that with the stripping protocol presented here the non-block technique of Western probing can be used for multiple sequential probing of the same blot with little or no loss of signal. This may be critical in cases where multiple probing of a valuable blot is necessary and maximal detection sensitivity is required. Acknowledgments. This work was supported by Grant RO1 AI26644 from the National Institutes of Health and by the Rosalind Russell Arthritis Center.
Documentation of Negatively Stained Polyacrylamide Gels 1 Terry M. Bricker,* Kari B. Green-Church,† Patrick A. Limbaugh,† and Laurie K. Frankel* *Department of Biological Sciences, Division of Biochemistry and Molecular Biology, and †Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803 Received November 16, 1999
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Mansfield, M. A. (1995) Anal. Biochem. 229, 140 –143. Stott, D. I. (1989) J. Immunol. Methods 119, 153–187. Laemmli, U. K. (1970) Nature 227, 680 – 685. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. Sadra, A., Cinek, T., Arellano, J. L., Shi, J., Truitt, K. E., and Imboden, J. B. (1999) J. Immunol. 162, 1966 –1973. Schneider, H., Cai, Y. C., Prasad, K. V., Shoelson, S. E., and Rudd, C. E. (1995) Eur. J. Immunol. 25, 1044 –1050. Sutherland, C. L., Krebs, D. L., and Gold, M. R. (1999) J. Immunol. 162, 4720 – 4730. Suck, R. W. L., and Krupinska, K. (1996) BioTechniques 21, 418 – 422.
Heavy metal negative staining (1, 2) is becoming increasingly popular for the detection of proteins separated by SDS–polyacrylamide gel electrophoresis. Advantages of the method include ease of use (rapid staining with destaining not being required), high sensitivity (intermediate to that exhibited by Coomassie blue and silver staining), reversibility (3), compatibility with Western blotting and subsequent immunodetection (4, 5), and 1 Funding was provided by the generous support of the National Science Foundation and the Department of Energy to T.M.B. and L.K.F. and the National Institutes of Health to P.A.L.
Analytical Biochemistry 278, 237–239 (2000) doi:10.1006/abio.1999.4446 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
238
NOTES & TIPS
compatibility with mass spectrometric procedures (6). Generally, SDS–polyacrylamide gels are soaked for a brief time in the chloride or sulfate salts of copper or zinc. The heavy metal salt forms an insoluble precipitate with the SDS. In some protocols the gels are pretreated with 200 mM imidazole (2), which provides a more homogeneous background. In all of these methods, the protein bands appear as clear zones embedded in a milky white background. While this method is becoming increasingly popular, the photographic documentation of gels stained in this manner is problematic. The gels inherently exhibit low contrast and are usually photographed with indirect lighting against a black background. It is often difficult to achieve even lighting across the entire gel, which leads to an apparent unevenness in the light-scattering of the background and poor image quality. Transmitted light photography, which is invariably used to document Coomassie blue-stained or silver-stained polyacrylamide gels, is inappropriate for use with negatively stained gels because the background transmits scattered light efficiently, leading to unacceptably low contrast images. In our laboratory, we have found that these gels can be easily documented by the use of a low-cost flatbed digital scanner. These devices utilize reflected rather than transmitted light to acquire an image. The milky white background of the negatively stained gels efficiently back-scatters light while the clear protein bands do not. When scanned with a black background (which is standard with most flatbed scanners), the protein bands appear dark against a white background. To demonstrate the utility of this documentation method, varying concentrations of standard proteins (Sigma Chemical Corp., SDS-6H) were separated on a 10% polyacrylamide gel using the buffer system of Laemmli (7). The proteins were electrophoresed overnight at 0.5 W. After electrophoresis, the gel was rinsed briefly in deionized water, and immersed in 300 mM CuCl 2 with gentle shaking for about 5 min. After full development of the negative image, the gel was rinsed briefly in deionized water and placed directly on the glass bed of a Microtek Scanmaker E6 digital scanner. The wet gel was overlayed with a piece of transparent acetate and the lid of the scanner, which contains the black background, was placed gently over the gel. The gel was scanned at 600 ⫻ 600 dpi resolution and either 256-level gray scale or 24-bit color resolution. Data acquisition times were 90 s for gray scale and 260 s for color. The image was acquired with PhotoImpact, Ver. 3.0 (Ulead Systems, Inc.) software and saved as a tagged-information format (tif) file. Contrast and brightness levels were adjusted to yield an image which closely corresponded to the visual appearance of the negatively stained polyacrylamide gel. For publication, the gel image was printed on an Epson 900
FIG. 1. Molecular weight standards separated by SDS–PAGE and copper-stained. After staining, the gel was scanned as described in the text, imported into Corel Draw 8.0 for labeling, and printed with an Epson 900 ink jet printer. The amount of standard protein in each protein band is shown above, while the molecular masses of the standard proteins are shown to the right.
ink jet printer on Kodak glossy ink jet paper after labeling with Corel Draw 8.0 (Corel Corp.). The results of this method of gel documentation are shown in Fig. 1. The image was captured using 256 gray scale levels and yielded an image file of 7 MB. The protein bands, which are transparent, do not efficiently scatter the light from the bulb of the digital scanner and appear as dark bands while the SDS– copper precipitate does back-scatter the light from the scanner and appears milky white. The image contrast is quite good, with the protein bands clearly visible in the scanned image. The results obtained with 24-bit color were comparable to the gray-scale image, exhibiting subjectively slightly improved resolution (data not shown). This slight image improvement was offset by a much larger image data file (18 MB) and consequently slower image processing times. While the results obtained from this method are clearly suitable for publication, it should be noted that the primary intent of this communication is to provide a simple, quick, and inexpensive method for the day-to-day documentation of negatively stained polyacrylamide gels. For most purposes, Coomassie blue staining or silver staining, followed by classical photography or digitization and image processing, provides superior images for publication. It should be noted that negative staining procedures have also been developed for use in detecting nucleic acids separated in either agarose or polyacrylamide gels (8). Our procedure should also be useful for the documentation of gels stained by these methods.
NOTES & TIPS
REFERENCES 1. Lee, C., Levin, A., and Branton, D. (1987) Copper staining: a five-minute protein stain for sodium dodecyl sulfate–polyacrylamide gels. Anal. Biochem. 1, 308 –312. 2. Fernandez-Patron, C., Castellanos-Serra, L., and Rodriguez, P. (1992) Reverse staining of sodium dodecyl sulfate–polyacrylamide gels by imidazole–zinc salts: Sensitive detection of unmodified proteins. Biotechniques 12, 564 –573. 3. Hardy, E., Santana, H., Hernandez, L., Fernandez-Patron, C., and Castellanos-Serra, L. (1996) Recovery of biologically active proteins detected with imidazole–sodium dodecyl sulfate–zinc (reverse stain) on sodium dodecyl sulfate gels. Anal. Biochem. 240, 150 –152. 4. Tessmer, U., and Dernick, R. (1989) Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate– polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of specific antisera. Electrophoresis 10, 277–279. 5. Wang, D., Dzandu, J. K., Hussain, M., and Johnson, R. M. (1989) Western blots from sodium dodecyl sulfate–polyacrylamide gels stained by metal salts. Anal. Biochem. 180, 311–313. 6. Cohen, S. L., and Chait, B. T. (1997) Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate–polyacrylamide gels. Anal. Biochem. 247, 257–267. 7. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680 – 685. 8. Hardy, E., Pupo, E., Casalvilla, R., Sosa, A. E., Trujillo, L. E., Lopez, E., and Castellanos-Serra, L. (1996) Negative staining with zinc–imidazole of gel electrophoresis-separated nucleic acids. Electrophoresis 17, 1537–1541.
Use of Hydrophobic Interaction Chromatography to Separate Recombinant Antibody Fragments from Associated Bacterial Chaperone Protein GroEL Kevin C. O’Connor,* ,1 Shibnath Ghatak,† and B. David Stollar* ,2 *Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, and †Division of Hematology/Oncology, Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts 02111 Received December 8, 1999
Many systems for expression of recombinant antibodies and antibody fragments have been developed in 1 Current address: Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Institutes of Medicine, Room 780, 77 Avenue Louis Pasteur, Boston, MA 02115. 2 To whom correspondence and reprint requests should be addressed at Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. Fax (617) 6362409. E-mail: [email protected].
Analytical Biochemistry 278, 239 –241 (2000) doi:10.1006/abio.1999.4465 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
239
the past decade. These systems enable rapid investigation into contributions of individual heavy and light chain V regions or particular amino acid residues to antigen recognition and binding. Whole antibody molecules have been expressed in plasmid-transfected mammalian (1) or baculovirus-infected insect cells (2), and Fab or Fv fragments have been expressed in yeast (3) or bacteria (4, 5). Bacterial systems have been especially popular because of their ease of genetic manipulation, efficient transformation, quick growth, and low cost. They have been used for production of Fabs (4), two-chain Fv (6), or scFv 3 (5, 7) domains, including those of both autoantibodies (8, 9) and immunizationinduced antibodies to DNA (10). We constructed plasmids pIg16 and pIg20 for the expression of scFv, VH, or VL domains fused to a single B domain of staphylococcal protein A (SPA) (10 –12). The vectors, derived from pGEMEX-1 (Promega, Madison, WI), use a T7 RNA polymerase promoter to drive transcription. The host cell is the BL21(DE3)–pLysE strain of Escherichia coli, engineered so as to contain the T7 RNA polymerase gene under lac promoter control (13, 14). Upon induction with IPTG, T7 RNA polymerase is produced and transcribes the pIg vector insert from the T7 promoter. A constitutively expressed plasmid pLysE, with a chloramphenicol resistance marker, is also present in these BL21 cells to ensure tight control of polymerase activation; a small amount of polymerase that may be formed before induction is bound and inactivated by the lysozyme produced from pLysE (13). Secretion of the expressed protein coded in the pIg vectors results from the presence of a bacterial alkaline phosphatase (Pho A) leader peptide coded at the 5⬘ end of the construct. In most cases, recombinant antibody– SPA fusion proteins are readily purified from clarified bacterial growth medium with an affinity chromatography column bearing IgG, to which the proteins bind through their SPA domains. The scFv–SPA, VH–SPA, or VL–SPA usually gives a single band on a Coomassie blue-stained SDS–PAGE gel (15). Sometimes, however, a bacterial protein copurifies with the antibody fragment. We have identified the bacterial protein as GroEL and have tested methods for separating the antibody fragment from it. We studied four preparations that contained bacterial protein along with scFv–SPA after they were purified from culture supernatants by capture on IgG– Sepharose (Pharmacia, Piscataway, NJ). They were derived from monoclonal antibodies dC1, dC5 (both anti-poly(dC)), 5E3 (ligand unknown), and SG45 (antiCD45). Migration of each of the scFv domains on SDS– 3 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; IPTG, isopropylthiogalactoside; scFv, single chain Fv domain.