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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

ISOLATION AND RENATURATION OF PROTEINS FROM SDS-PAGE GELS

Isolation and Renaturation of Proteins from SDS-PAGE Gels
Contributor: The Laboratory of Jim Kadonaga at the University of California, San Diego
 
Overview
This protocol describes a method to purify a protein through an SDS-polyacrylamide gel and then elute the protein from the gel. The protein is then Acetone precipitated and renatured with Guanidine. The protocol is adapted from Citation #1. The contributor of this protocol suggests that users refer to this paper for excellent additional information before attempting a renaturation experiment. Citation #2 is another useful resource.
 
Procedure
A. Preparation of Polyacrylamide Gel

1. Pour an SDS-PAGE Running Gel of the desired percentage according to the recipe provided or an SDS-Polyacrylamide Gel recipe of choice (see Hint #1).

2. Layer with 2-Butanol.

3. After the Running Gel has polymerized, pour off the 2-Butanol and rinse with 0.1% SDS.

4. Layer 0.1% SDS on the Running Gel and cover the gel with plastic wrap.

5. Allow the gel to polymerize overnight at room temperature.

6. Pour off the 0.1% SDS from the running gel.

7. Prepare the Stacking Gel with the recipe provided or an SDS-Polyacrylamide Stacking Gel recipe of choice.

8. Allow the Stacking Gel to polymerize for at least 1 hr but not more than 2 hr.

B. Preparation and Electrophoresis of the Protein Sample (see Hint #2)

1. Calculate the amount of sample that needs to be loaded in order to observe activity after renaturation. Assume a poor yield from the gel of approximately 10%.

2. Prepare a sample to be loaded on the gel and a mock sample that will not be loaded on the gel but will be taken through all other manipulations.

3. Add 2 volumes of 2X Sample Buffer to both the sample and the mock sample.

4. Incubate both samples at 65°C for 15 min.

5. Freeze the mock sample at -20°C until Step #E1.

6. Load the sample and molecular weight markers on the gel.

7. Electrophorese the sample (see Hint #3).

C. Visualization of the Protein in the Gel Using Salt Stain (see Hint #4)

1. Rinse the gel with ddH2O.

2. Stain the gel by soaking the gel in ice-cold KCl/DTT for 5 min.

3. If required, destain the gel in cold 1 mM DTT.

4. The staining is best observed with oblique light against a black background. The proteins should appear as faint white bands.

5. Cut out the desired band with a razor blade or scalpel blade, keeping the size of the gel slice to a minimum.

D. Elution of the Protein from the Gel Slice

1. Put the gel slice into a microcentrifuge tube if possible.

2. Wash the gel slice twice with ddH2O.

3. Soak the gel slice in 1 mM DTT for 15 min.

4. Homogenize the gel slice with a Teflon coated Eppendorf© homogenizer.

5. Add several volumes of Elution Buffer.

6. Incubate the gel slice in Elution Buffer for 1 to 12 hr with occasional mixing (see Hint #5)

7. Centrifuge in a microcentrifuge briefly to pellet the gel debris.

8. Remove the supernatant carefully while avoiding the pieces of Polyacrylamide Gel. Place the supernatant in a siliconized 15 ml glass Corex tube.

E. Removal of SDS from the Protein and Acetone Precipitation of the Protein

1. Thaw the mock sample.

2. Add Elution buffer to the mock sample so that it has a volume equal to the supernatant collected in Step #D8.

3. Add 5 volumes of -20°C Methanol:Acetone to both the mock sample and the eluted protein sample. Vortex gently.

4. Incubate both tubes at -20°C overnight.

5. Chill a Sorvall SS34 rotor and the centrifuge chamber to -5°C.

6. Centrifuge the samples at 10,000 rpm in an SS-34 rotor (12,000 X g) for 15 min at -5°C.

7. Carefully remove the supernatants with a drawn-out Pasteur pipette to remove the last bit of supernatant.

8. Gently add 1 or 2 ml of Methanol:Acetone to rinse the pellets. DO NOT vortex.

9. Centrifuge the samples at 10,000 rpm in an SS-34 rotor (12,000 X g) for 15 min at 4°C.

10. Carefully remove the supernatant.

11. Air dry the protein pellets on ice.

F. Guanidine HCl Renaturation of the Protein (see Hint #6)

1.Gently resuspend the protein pellet in the minimum possible volume of Buffer A.

2. Incubate at room temperature for 15 to 20 min.

3. Add several volumes of Buffer B.

4. Dialyze at room temperature for 2 to 3 hr against Buffer B in a Microdialyzer (Pierce).

5. Freeze the protein solution in Liquid Nitrogen and store at -80°C.

6. Use an assay of choice to assess the enzymatic activity of the protein.

Solutions
Buffer B   1 mM DTT
0.1 mM KCl
0.01% (v/v) IGEPAL CA-630
12.5 mM MgCl2
10% (v/v) Glycerol
25 mM HEPES-KOH, pH 7.6
0.1 mM EDTA
Buffer A   100 mM KCl
1 mM DTT
12.5 mM MgCl2
6 M Guanidine
10% (v/v) Glycerol
25 mM HEPES-KOH, pH 7.6
0.1 mM EDTA
Lower Tris Buffer (4X)   Add ddH2O to 500 ml
Adjust pH to 8.8 using HCl
90.85 g Tris base
20 ml of 10% SDS (C12 Grade; Pierce)
Store at 4°C
HEMG   1 mM DTT
12.5 mM MgCl2
10% (v/v) Glycerol
25 mM HEPES-KOH, pH 7.6
0.1 mM EDTA
Stacking Gel   5% of 37.5:1 Acrylamide:Bisacrylamide (CAUTION! See Hint #8)
0.1% (w/v) SDS (C12 Grade; Pierce)
0.125 M Tris-Cl, pH 6.8
10 μl TEMED per 10 ml of solution
Just before pouring gel add:
50 μl 10% Ammonium Persulfate per 10 ml of solution
Methanol:Acetone   1:1 Methanol:Acetone
SDS-PAGE Running Gel   X% Acrylamide:Bisacrylamide should be determined based on the size of the protein of interest (see Step #A1).
28 μl 10% Ammonium Persulfate per 10 ml of solution
0.1% (w/v) SDS (C12 Grade; Pierce) (See Hint #7)
0.375 M Tris-Cl, pH 8.8
X% of 37.5:1 Acrylamide:Bisacrylamide (CAUTION! See Hint #8)
6 μl TEMED per 10 ml of solution
Just before pouring gel add:
1 mM DTT
10% Ammonium Persulfate   10% (w/v) Ammonium Persulfate
Store at 4°C for 3 weeks
KCl/DTT   1 mM DTT
0.25 M KCl
Elution Buffer   5 mM DTT
50 mM Tris-Cl, pH 7.9
0.1% SDS (C12 Grade; Pierce)
0.15 M NaCl
0.1 mM EDTA
0.1% SDS   0.1% (w/v) SDS (C12 Grade; Pierce)
Sample Buffer (2X)   2% (v/v) 2-Mercaptoethanol
10% (v/v) Glycerol
0.1% (w/v) Bromophenol Blue
4% (w/v) SDS (C12 Grade; Pierce)
100 mM Tris-Cl, pH 6.8
Add just before use:
Upper Tris Buffer (4X)   pH to 6.8 using HCl
Add ddH2O to 100 ml
4 ml of 10% SDS (C12 Grade; Pierce)
6.06 g of Tris base
Store at 4°C
0.1 mM Thioglycolate (see Hint #9)
 
BioReagents and Chemicals
Magnesium Chloride
Glycerol
2-Mercaptoethanol
IGEPAL CA-630
Thioglycolate
Acetone
Potassium Hydroxide
Tris
Potassium Chloride
Sodium Chloride
DTT
2-Butanol
Nitrogen, Liquid
Bromophenol Blue
Methanol
HEPES
SDS, C12 Grade
EDTA
Acrylamide:Bisacrylamide
TEMED
Ammonium Persulfate
Guanidine
 
Protocol Hints
1. Remember that proteins will elute more quickly and more efficiently from a low percentage gel.

2. TCA precipitation of the sample is not recommended, if it can be avoided.

3. For Bio-Rad mini-gel system, run the samples at 8 mAmps constant current through the stacking gel and at 18 mAmps constant current through the running gel.

4. This method of staining is not very sensitive. You need to load a great deal of your protein to be able to see it. If that is not feasible, consider staining with Coomassie Blue, which has been reported to work reasonably well with renaturation.

5. The exact elution time will depend on the molecular weight of the protein. Generally, larger proteins will require longer incubation times.

6. The authors of Citation #1 recommend suspending the protein pellet in 6 M Guanidine, diluting the protein sample 50-fold, and then incubating it at room temperature before assaying the activity of the protein. A 50-fold dilution may or may not suffice to reduce the Guanidine concentration for the purposes of the assay of choice. Additionally, a 50-fold dilution may dilute the protein too much for it to be used to assay its activity. The contributor of this protocol dilutes the sample several fold and then dialyzes the protein solution to minimize the dilution necessary while reducing the Guanidine concentration.

7. Use the best quality SDS available for all buffers, sample buffers, and running buffers that call for SDS.

8. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

9. The Thioglycolate will scavenge the gel for any remaining free radicals, preventing them from damaging the protein.

 
Citation and/or Web Resources
1. Hager DA and Burgess RR. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980;109:76-86
2. Hunkapiller MW, Lujan E, Ostrander F, Hood LE. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983; 91:227-36.