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Indian Journal of Biochemistry & Biophysics

Vol 44, April, 2007, pp. 122-125

Note

Purification of protein from a crude mixture been reported4. In all the above studies, attempts have
through SDS-PAGE transfer method been made for identification and subsequent isolation
after a single run, where contamination from quite
Dipankar Bhattacharyya, Arindam Basu and closely separated proteins is possible.
Parimal C Sen* Here, we report a procedure for protein
Department of Chemistry, Bose Institute, 93/1, A.P.C. Road, purification/separation and enrichment using
Kolkata 700 009, India transfer method of SDS-PAGE gel. We have found
Received 06 July 2006; revised 27 February 2007 that for higher recovery of protein, ZnSO4 staining
was more effective, as compared to Coomassie blue
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) transfer staining procedure. Elution parameters have been
method was used for purification and enrichment of the protein optimized for maximum possible recovery (70%),
from crude sample. Coomassie blue/ZnSO4 stained protein when ZnSO4 is used as staining agent. In the
band(s) containing intact polyacrylamide gel were loaded on to
another polyacrylamide gel either alone or as pooled gel bands.
method, for the first time, a protein band has been
Two/three bands were combined together and arranged tightly transferred from first to second gel for further
over one another, sealed with stacking gel and ran in another gel, purification from possible contamination of closely
which was quite useful for enrichment and purification of a separated protein(s). Enrichment of a particular
particular protein from a complex mixture. Recovery of protein by
protein with improved purity has been acheived by
gel transfer method was found to be 70% in case of ZnSO4
staining, whereas around 30% recovery was possible, following collecting multiple bands of same protein from first
Coomassie blue staining. The method described here for gel, followed by transfer and rerun in the second
purification of protein(s) from a complex mixture, following gel gel.
transfer procedure could be useful for further characterization of
the desired protein. Materials and Methods
Materials
Keywords: SDS-PAGE, Coomassie blue and ZnSO4 staining, Acrylamide, bis-acrylamide, glycine, imidazole,
Gel transfer, Protein purification
ammonium persulphate, glycerol, NaCl and Tris-HCl
In electrophoresis, the rate of migration of charged were purchased from SRL Laboratory, Mumbai,
molecules depends on the strength of the field, net India; ZnSO4 from Merck, India, sodium
charge, size and shape of the molecules and also on dodecyl sulphate, bovine serum albumin (BSA),
the ionic strength, viscosity and temperature of the N,N,N,N tetramethyleneethylenediamine (TEMED)
medium. As analytical tool, electrophoresis is simple, and -mercapto ethanol (ME) from Sigma
rapid and highly sensitive and is used to study the Chemicals Co., St. Louis, MO, USA, and Mini gel
properties of a single charged species and as a casting apparatus was from Technolab, Kolkata.
separation technique. SDS-PAGE is generally used to Methods
separate proteins, depending on their electrophoretic Quantification with known protein
mobility, due to differences in charge1. SDS-PAGE, SDS-polyacrylamide gel (10%) was prepared
followed by electroelution is a well-known technique2 following standard procedure. In one gel, known
for protein isolation/purification, but the protein is amounts (5, 10 and 15 g each) of BSA were run and
obtained in an extremely diluted form, thus requires gel was stained with Coomassie blue or ZnSO4.
concentration of the extract. Recently, imidazole- Stained bands were cut from the gel and transferred to
ZnS04 staining has been described for detection of another 10% gel either as a single or overlay
protein after gel electrophoresis3. Subsequently, (double/triple layering) and sealed. The gel was run as
advantages of imidazole-sodium dodecyl sulphate- before followed by staining and destaining either with
ZnS04 reverse staining in polyacrylamide gels have Coomassie blue or ZnSO4 method. Coomassie or
______________ ZnSO4 stained bands were scanned for quantitation of
*Author for correspondence
Fax: (+91) 33 2350 6619 each protein using Bio-Rad (Model GS-700) Imaging
Email: senpc03@yahoo.com; parimal@bosemain.boseinst.ac.in Densitometer. Similarly, the experiment was repeated
NOTES 123

with a higher and fixed amount (50 g) of BSA and as given above and left for overnight. The suspension
quantitation was made as described above. containing gel pieces was subjected to
homogenization. The mixture was taken in Eppendorf
Purification of protein from a mixture vials and spun at 8,000 g at 4C. Supernatant was
Two sets of 10% SDS-polyacrylamide gel were collected, pellet was again subjected to
prepared. In one gel, goat testes cytosolic fraction homogenization with 2 ml elution buffer and
containing a mixture of 116 kDa (protein kinase-x)5 centrifuged as above. Supernatant was collected and
and 70 kDa (an inhibitor of Na, K-ATPase)6 proteins the procedure was repeated again. Protein in the
was run. The gel was stained, destained with supernatant was estimated using Bradford method8.
Coomassie blue and bands corresponding to two
proteins were isolated and each loaded on to another Results and Discussion
gel separately. The second gel was stained and In this study, gel transfer method was used to
destained as described before. Bands were quantified purify proteins from a complex mixture. Fig. 1A
with a densitometric scanner. shows the SDS-PAGE of known amount of BSA. The
Enrichment of specific protein gel was scanned densitometrically, followed by
A specific protein of interest (70 kDa or 116 kDa) second run (Fig. 1B) and stained with Coomassie
in a mixture of crude sample from goat spermatozoa blue. Similarly, Figs 1A and B show corresponding
was run in a gel. Coomassie blue/ZnSO4 stained densitometric scanned gels stained with ZnSO4.
band(s) containing the protein of interest (above) was Fig. 1A shows that in case of ZnSO4 although
cut off from the gel and combined together and loaded absorbance of each band of stained gels was more or
double/triple layered in the second gel, sealed with less comparable, but was lower than that of
stacking gel and rerun. Two proteins of molecular Coomassie blue stained bands. The reason could be
mass 116 and 70 kDa present in the cytosolic fraction due to absorption of dye by protein in this case,
of goat testes were purified/enriched following gel whereas in case of ZnSO4 stained bands were
transfer method. Second gel was stained with completely transparent. However, when absorbance in
Coomassie blue/ZnSO4, followed by densitometric Fig. 1B (1, single, 2, double or 3, triple band slices)
scaning. Proteins were eluted from the gel by was compared with Fig. 1A, recovery was found to
homogenizing with the elution buffer (20 mM Tris, be about 70%.
pH 7.5-7.8, 150 mM NaCl, 2 mM ME and 10%
glycerol).
ZnSO4 staining of proteins
To achieve higher sensitivity or efficient
mobilization of proteins after staining, a rapid
reversible ZnSO4-imidazole staining method was
used7. After run was complete, gels were incubated
for 10-15 min in 0.2 M imidazole, 0.1% SDS and
rinsed with distilled water. Proteins were visualized as
transparent bands against milky background by
soaking the gel in 0.2 M ZnSO4 for 1-2 min. Fig. 1(A): 10 g BSA was loaded on to each lane (1-6) and
Destaining and mobilization of proteins were 10% SDS-PAGE was performed as described in Materials and
Methods; and (B): Bands from lanes in (A) were cut and loaded
accomplished by incubating the gel in 50 mM Tris, on to gel as a single (lane 1), two (lane 2) and three (lane 3) bands
192 mM glycine, 0.1% SDS, 50 mM EDTA together. Electrophoresis was performed as described in
(mobilization buffer, MB) until background was Materials and Methods, followed by staining and destaining
completely clear. Protein was estimated by Bradford with Coomassie blue. (A): 10 g BSA was loaded on to each lane
method8 which showed 70% recovery. (1-6) and 10% SDS-PAGE was performed as described in
Materials and Methods; and (B): bands from lanes in (A) were
Protein elution from SDS-polyacrylamide gel cut and loaded on to gel as a single (lane 1), two (lane 2) and three
(lane 3) bands together. Electrophoresis was performed as
Designated band(s) from the gel was cut into pieces
described in Materials and Methods, followed by staining and
with a scalpel blade, suspended and minced into small destaining with ZnS04. Results shown here were one
pieces in a small volume (~2 ml) with elution buffer representative from three separate experiments in each case
124 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, APRIL 2007

Table 1 Mean absorbance from densitometric scan of Coomassie blue and ZnS04 stained bands from Figs. 1A, B, A, B
[Values were mean S.D. of 3 different experiments]
Lane 1 2 3 4 5 6
Coomassie blue stained (Fig. 1A)
Mean absorbance 0.317 0.014 0.330 0.016 0.293 0.012 0.334 0.014 0.328 0.009 0.342 0.015
Commassie blue stained (Fig. 1B)
Mean absorbance 0.095 0.015 0.193 0.013 0.308 0.021
% of Transfer 30.5 2.9 31.2 2.4 29.9 3.0
ZnS04 stained (Fig. 1A)
Mean absorbance 0.056 0.013 0.060 0.009 0.062 0.012 0.060 0.015 0.055 0.008 0.044 0.010
ZnS04 stained (Fig. 1B)
Mean absorbance 0.040 0.050 0.084 0.016 0.116 0.020
% of Transfer 69.8 5.5 72.5 6.8 71.4 4.7
Table 2Mean OD from densitometric scan of Coomassie blue
stained bands of unknown proteins from Figs 2 A, B and C
[Results were mean S.D. of 3 independent experiments]
Fig. 2A
Lane 1 Lane 2 Lane 3
Mean OD
116 kDa band 0.054 0.005 0.057 0.006 0.056 0.006
70 kDa band 0.352 0.010 0.360 0.09 0.334 0.011
Fig. 2(A): 10% SDS-PAGE of a protein mixture isolated from
goat testes cytosol. Lanes 1, 2 and 3 each contains 100 g protein. Fig. 2B
M, molecular weight markers (116 and 66 kDa); (B): 116 kDa Mean OD 0.017 0.004 0.033 0.008
(PKx) protein band from lane 1 alone and lanes 2 and 3 combined % of Transfer 31.5 3.88 29.8 2.95
from (A) was loaded on to lanes 1 and 2 respectively; and (C): 70 Fig. 2C
kDa (inhibitor protein) band from lane 1 alone and lanes 2 and 3 Mean OD 0.103 0.014 0.224 0.017
combined from (A) was loaded on to lanes 1 and 2 respectively. % of Transfer 29.2 2.63 32.3 1.28
Results shown were one representative from three separate
experiments in each case
and the Coomassie blue stained bands on 10% SDS-
Results (absorbance) from densitometric scanning PAGE are shown in the Figs 2A, B and C. Both 70 and
of Coomassie blue stained bands from Figs. 1A and B 116 kDa bands in Fig. 2A, 116 kDa in Fig. 2B and
are shown in Table 1. It was evident from the Table 70 kDa in Fig. 2C were scanned densitometrically and
that absorbance of each band of BSA corresponds to quantitated in each case as shown in Table 2. It was
lanes 1-6 in Fig. 1A was more or less comparable. evident from the densitometric scaning results of Figs
Staining of gels with ZnSO4 before Fig. 1A and after 2A, B and C that transfer of both single and double
Fig. 1B transfer (one, two or three slices) suggested bands of 116 and 70kDa proteins was almost identical
concentration-dependent transfer (Table 1), which (around 30%) as shown in Table 2. These findings
was much better than Coomassie blue staining were similar to the recovery of BSA (Table 1,
method. Absorbance in Table 1 corresponds to corresponding to Fig. 1B). Protein(s) after transferring
Fig. 1B both from single (lane 1), double (lane 2) and could either be concentrated or enriched by loading
triple (lane 3) slices, the recovery was found to multiple bands together from the preceding gel run.
be around 30% and reproducible. This low Even with higher concentration of BSA (50 g),
recovery/transfer might be due to fixing of gels during recovery was found to be 30% with Commassie blue
Coomassie staining, leading to the poor diffusion and and 70% with ZnSO4 staining (Table 3).
thus transfer was not the same into the second gel as The SDS-PAGE transfer method may be useful for
was observed in case of ZnSO4 staining. In ZnSO4 isolation/purification of protein(s) from a mixture for
staining, almost 70% consistent transfer was possible (i) Study of functional aspect of proteins for
with one, two or three slices (Table 1). proteomic analysis, (ii) generation of antibodies, and
The purification of 70 kDa and 116 kDa proteins (iii) sequencing etc. Some advantages of the described
from goat testes cytosol through gel transfer method method are: (a) Protein could be purified from crude
NOTES 125

Table 3Recovery of protein (BSA) after staining first gel which may cause problem in diffusion of protein from
with Coomassie blue or ZnSO4, followed by rerun in the the gel slices.
second gel
In conclusion, the SDS-PAGE transfer method
[Values were mean S.D. of 3 different experiments] described here is useful for isolation/purification of a
Staining of 1st Protein excised from % Recovery desired protein from a complex/crude mixture for
gel unfixed 2nd gel (g) further studies as described above.
Coomassie blue
50 g 15.2 1.84 30.3 3.68 Acknowledgement
ZnSO4 The work was supported from the financial aid
50 g 35.1 2.74 70.3 5.45 from Council of Scientific and Industrial Research,
New Delhi (37 (1088)/01-EMRII) and Bose
sample containing intact polyacrylamide gel, followed Institute, Kolkata.
by electrophoretic rerun in second gel, without eluting
the protein; (b) enrichment of a particular protein References
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6 Mandal A K, Roy K, Sil P C, Yadav S P & Sen P C (2001)
However, the method has the following limitations: Mol Cell Biochem 223, 7-14
(i) Excess loading of protein was not possible due to 7 Fernandez-Patron C, Calero M, Rodriguez-Collazo P, Gracia
J R, Madrazo J, Musacchino A, Soriano F, Estrada R, Frank
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many gel slices could not be loaded on to second gel, 8 Bradford M M (1976) Anal Biochem 72, 248-254

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