Detection of basic proteins and low molecular weight peptides in polyacrylamide gels by formaldehyde fixation

Detection

Weight Pep ltides in Fixation

of Basic Proteins and Low Molecular Polyacrylamide Gels by Formaldehyde GERMAINE

STECK,’

PETER

LEUTHARD.’

AND

ROBEKI

R.

BUKK

Fricdric~h !~lr~~.\~~/~t~~-l,~.,rirlrr. P.0. Bt,.v-773. C‘H-400_7 BCI\d. J ~~~ir,crl~rtr~l Received Certain

basic

and

fixation procedures. tides in polyacrylamide proteins.

low

molecular

Formaldehyde gels. The

In all the systems

tested

weight has method the new

method and also additional low molecular ampholines which were lost in the standard the detection of these substances.

November

26.

proteins

are

heen used has been method

1979 not

retained

to covalently tested with

retained

in gels

hy standard

acid

link proteins and polypepa variety of gel system:, and

all the proteins

weight and basic proteins systems. Therefore. the

seen

in the standard

and polypeptides method is suitable

and for-

Staining methods presently utilized for hyde-fixed proteins on polyacrylamide gels localizing proteins in acrylamide gels require with Coomassie Brilliant Blue R-250 stainprior denaturation of proteins in acid (I]. ing which were not otherwise detectable. followed by staining and removal of excess dye. Classically. proteins are precipitated MATERIALS AND METHODS with acetic acid, trichloroacetic acid, or dlftrtc~ricrls sulfosalicylic acid and combinations of all or some of these and/or by various alcohols. Acrylamide, N.N’-methylenebisacrylWe found that certain basic and low mo- amide were from Bio-Rad Laboratories lecular weight proteins remain soluble under (Richmond. Calif.). All other reagents were obtained from Merck (Darmstadt, West these acid conditions or. if precipitated. are readily solubilized again during the Germany]. staining or destaining of the polyacrylStandard proteins were analytical grade amide gel. from Serva (Heidelberg, West Germany): It occurred to us that proteins could be bovine serum albumin, M, 68.000, pl 4.7: retained in gels by fixing them there chem- pepsin, M, 36,000. pf 2.2; chymotrypsinogen ically as opposed to the usual physical A. M, 25,000. pl 9.1; lysozyme, M, 14.400. denaturation. Since formaldehyde forms p1 11.35; ribonuclease A, M, 13.600. pl 9.3: methylene bridges between amino groups protamine sulfate. on the one hand and primary amide or Hormones were synthetic: h-calcitonin, guanidyl groups on the other (2). we in- il4,. 3318: substance P, M, 1338: h-ACTH,’ vestigated it as a possible means of cross- XI, 4541; (r-MSH, M, 1725: somatostatin. linking polypeptides to polyacrylamide. M,. 1638; LHRH, M,. 1182. In this paper we describe a procedure All hormones were kindly provided by Dr. which allowed US to detect bands of formalde- A. Joehl. Ciba-Geigy, Basal. Switzerland. ’ .4hhreviations ’ Present address: ceutical Division. Basel. Switzerland.

Research Ciba-Geigy

Department. Limited.

PharmaCH-4002

used:

wMSH, ct-mclanotropin. sodium dodecyl sulfate: meth~lrth~lenediamine.

h-ACTH. LHRH. Temed.

human

corticotropin;

luliherin: SDS. ~~.~~..V’.,~‘-tctr~t-

22

STECK.L,ElJTHARD,ANDBiTRK

Methods

Polyucrylcrmide gel electrophorrsis. All gels were cast as slabs in glass formers. The gels were run in vertical electrophoresis equipment (Biotec AG, Schonenbuch, Switzerland). Native gel systems used according to H. R. Maurer (1) were: No. 1 at pH 8.9,7.5% (w/v) acrylamide; No. 5 at pH 7.3. 11.0% (w/v) acrylamide; No. 7 at pH 4.3, 15%~(w/v) acrylamide; No. 9 at pH 2.9, 11.0% (w/v) acrylamide. Urea gels correspond to the second dimension described by Ramjoue and Gordon (3). SDS gels correspond to the method of Lammli (4). Polyacrylamide gels (1.5% acrylamide containing 0.4% bisacrylamide) were made in 0.1% SDS-O.375 M Tris-HCl at pH 8.8. SDSgels were polymerized with 19 mg ammonium persulfate and 50 ~1 Terned/ ml acrylamide solution. Running buffer was 0.025 M Tris, 0.2 M glycine. and 0.1% SDS. Stcrining methods. Two staining methods were used: Method 1 is the technique described by LKB (5) which uses denaturation. fixation, and staining in acid. Method 2 is the optimum we have found for formaldehyde fixation. Both methods were always performed in a fume cupboard. Method 1. 1. Proteins were denatured by immersing the gel in “fixing” solution (containing 17.3 g sulfosalicylic acid and 57.5 g trichloroacetic acid in 500 ml of distilled water) for 0.5 to 1 h. 2. Gels are placed in staining solution for 10 min at 60°C or 1 h at room temperature (0.46 g Coomassie Brilliant Blue R-250 in 400 ml “destaining” solution). 3. Excess dye is removed by immersing the gel in “destaining” solution (500 ml ethanol with 160 ml acetic acid made up to 2 liters with distilled water). Frequent changes of the solution are necessary and a clear background requires about 24 h destaining. Method ,1. 1. Proteins were fixed and stained at the same time by immersing the

gel for 1 h in a solution containing 180 ml ethanol, 420 ml distilled water, 100 ml 35%’ formaldehyde, and 0.8 g Coomassie Brilliant Blue R-250. la. Proteins were further stained for 1 to 3 h with a reduced formaldehyde concentration to avoid gel shrinkage in a solution containing 250 ml ethanol, 750 ml distilled water, 10 ml 35% formaldehyde and 1.2 g Coomassie Brilliant Blue R-250. 2. Gels were immersed in destaining solution containing 250 ml ethanol, 750 ml distilled water, and 10 ml 35%’ formaldehyde. Destaining is carried out overnight with several changes of solution at the beginning. Step la was added for SDS-gels. Protein bands stained with Coomassie Brilliant Blue were photographed under white light illumination, using a Polaroid type 665 film at f 32 and exposure time of 112s with a yellow filter. RESULTS Method 2 has been used successfully to stain proteins in each of the six systems listed in the Methods section. A standard mixture consisting of ribonuclease A, lysozyme, and chymotrypsinogen A was tested. Ribonuclease and especially protamine sulfate were easily washed out of the gel by the acid staining and destaining technique producing either a pale blue zone or a large diffuse dark blue zone. With longer destaining the protamine bands disappear altogether using method 1. In contrast. the formaldehyde method gave tight clear bands that were stable for up to 2 months in the destaining solution. The particular advantages of the new method for the detection of basic proteins (6) and peptides are illustrated here. We are purifying a highly basic protein (5) that can be expected to be contaminated with other basic proteins. Figure 1 shows a SDS-polyacrylamide gel with two different samplesof basic components of conditioned medium

PEPTIDE

AND

BASIC

PROTEIN

from cell cultures of SV28 cells (6). The gel was cut in two halves and stained with the two methods. Whereas in method 1 the protein channel B looks nearly pure, we can detect many low molecular weight substances in the extracts from SV28 cells (channels A + B) using method 2. In another step of the purification the new method allowed us to detect basic proteins from conditioned medium after fractionation on a Sephadex G-75 superfine column. Twenty micrograms of protein from each of five fractions from different molecular weight regions were analyzed on a SDSpolyacrylamide gel. Most of the protein was washed out of the gel using method I. whereas distinct bands could be seen with the formaldehyde method. In the analysis of basic proteins (Fig. I) it can be seen that the new method has particular advantages in detecting substances in the low molecular weight range. Therefore, we now illustrate this advantage with

DETECTION

IN

23

GEI,S

&mine ABCDEF

ABCDEF FI(,. LHRH.

2.

I>?

40 +g:

Polyacrylamide (B)

somatostatin,

gel.

pH

20 pg:

20 w*g: (1)) h-ACTH. 20 pg: tE) substance (F) h-calcitonin. 40 wg. The hormones were Protein migration from top to hottom.

(C)

7.3.

t.41

wMSH. P. 20 pg: synthetic.

defined peptides. Synthetic hormones were applied to a 15%’ polyacrylamide gel at pH 7.3. Again the gel was cut in two pieces and stained with the two methods. We found (Fig. 2) that the more basic polypeptides were washed out of the gel in the acid staining technique, but a good band was clearly visible with the formaldehyde method in each channel and further in some cases very minor components could be detected. DISCUSSION

A pH

B

FIG. I. IYc; Polyacrylamide 8.8. A + B each. 20 pg

conditioned Protein

A gel with of protein

medium from cell culture\ migration from top to bottom.

B O.l’i SDS of extracts of SV’78

cell\.

at ut

By a simple two-step procedure one can detect basic low molecular weight proteins and polypeptides. During optimization of the conditions used, formaldehyde concentration was varied between 1 and 10%. The concentration chosen was 5% because in a higher concentration the gel shrunk drastically and at lower concentrations fixation was not satisfactory. At this concentration I h was found to be adequate to fix proteins in the gels but not long enough to stain them in the presence of SDS. Therefore step la was added for SDS-gels. Although formaldehyde seems satisfactory

24

STECK.

LEUTHARD,

for analytical purposes, it is conceivable that other crosslinking reagents would have advantages (7), particularly. if the bond could be broken to allow the recovery of the biological activity. Enzyme activity has already been demonstrated in neutral formalinacetate-fixed gels (8). which gives the hope that biological activity may not, in every case, be destroyed by formaldehyde. Results with isoelectric focusing gel systems have not been satisfactory. Ampholines are also fixed by the formaldehyde crosslinking reaction and then stained by Coomassie Brilliant Blue R-250. Perhaps a different stain can be found that would avoid this problem. However, for urea. SDS-, and native polyacrylamide gel systems at a large pH range. this formaldehyde fixation linked to Coomassie Brilliant Blue staining represents an easy and effective procedure.

AND

BiiRK

ACKNOWLEDGMENT We

thank

Dr.

H.

Towbin

for

performing

the

urea

gel electrophoresia.

REFERENCES 1. Maurer.

H.

R.

(1971)

Related Techniques Electrophoresla. 2.

New York. Fraenkel-Cunrat.

3.

J. Amer. Ramjoue. J. Rio/.

4. 5 6. 7.

H.,

252.

Lammli. U. K. (1970) 680-68.5. Winter. A.. Ek. K.. and LKB Application Burk, R. R. (1976) Stark. G. Reactions

R. on

Electrophoresis

and

of

Polyacrylamide de Gruyter.

Gel Berlin/

Walter

C’iwt~. Sot. H. -P., R.. C‘hc~m.

Disc

and

Olcott.

H.

70, 1673. and Gordon.

(1948)

J.

( 1977,

‘9065-9070. Ntr/ltw

(L~j~zdr~!r/

Andersson.

U.

Note 150, December Erp. C‘c,// Kc\. 101, (1971) Solid

S.

Biochemical Support\,

Academic Press, New York. A. F.. Takakura. K.. and 8. Goldberg, R. L. (1966) Vtrtltrc (Lorrtlo,~) 211.

227,

A. (1977) 1977. 193-298.

Aspects of pp. 27-23. Rosenthal. 41-43.