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| This technique requires that the antibody recognizes the denatured antigen. Many antibodies will recognize the denatured antigen. The recovery of protein using this protocol is approximately 10 to 20% of the starting material. |
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A. Elution of Protein from Acrylamide Gel
1. Electrophorese the proteins on a SDS polyacrylamide gel (See Protocol on SDS Polyacrylamide Gel Electrophoresis) (See Hint #1).
2. Excise the bands representing the protein of interest from the polyacrylamide gel. (See Hint #2).
3. Dice the gel into small pieces.
4. Wet the gel pieces with a small amount of Elution Buffer.
5. Place the pieces in the bottom of a 15 ml or 30 ml glass centrifuge tube. (See Hint #3).
6. Add 3 ml of Elution Buffer. (See Hint #4).
7. Add 2-Mercaptoethanol to 5.0% (v/v).
8. Boil the mixture for 5 min.
9. Add 20 μl of the Bovine Gamma Globulin Solution. (See Hint #5).
10. Incubate the mixture for 18 hours at 37°C in a shaking water bath with vigorous agitation.
11. Remove the liquid and place the liquid in a 15 ml glass tube at 0°C.
12. Add 1 ml of Elution Buffer to the gel pieces and elute for 4 hr at 37°C in a shaking water bath with vigorous agitation.
13. Remove the liquid.
14. Pool the two eluates (from Step 11 and 13) and place them at 0°C.
B. Isolation of Protein from Eluate
1. Add 20 μl of the Bovine Gamma Globulin Solution.
2. Add 1 ml of 100% Trichloroacetic Acid.
3. Parafilm the top of the tube.
4. Vortex the tube to mix the contents.
5. Incubate at 0°C for 18 hr.
6. Centrifuge the mixture for 40 min at 16,400 X g at 0°C.
7. Carefully remove the supernatant with a Pasteur pipette. (See Hint #6).
8. Wash the pellet by adding 5 ml of 100% Ethanol (ice-cold).
9. Vortex gently to dislodge the pellet.
10. Centrifuge the mixture for 40 min at 16,400 X g at 0°C.
11. Carefully remove the supernatant using a Pasteur pipette. (See Hint #6).
12. Wash the pellet again (Repeat Steps #8 to #11).
13. After the second Ethanol wash, allow the tube to drain well by inverting the tube on a kimwipe.
14. Also carefully wipe the inside rim of the tube with a kimwipe.
C. Imunoprecipitation of Protein
1. Add 400 μl of the 2X Modified RIPA Buffer and transfer the mixture to an microcentrifuge tube.
2. Rinse the glass tube with 100 μl of the 2X Modified RIPA Buffer. Pool this Buffer with the Buffer from the previous step.
3. Add 500 μl of ddH2O to the microcentrifuge tube.
4. Let the mixture sit at 0°C for 5 to 10 min.
5. Once the mixture has clarified, transfer the upper clear layer to a new microcentrifuge tube. The antigen mixture is now ready for immunoprecipitation by the appropriate antibody. (See Hint #7).
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| Modified RIPA Buffer (2X) |
| 2% (v/v) NP-40
2% (v/v) Sodium Deoxycholate
4 mM EDTA
20 mM Tris, pH 7.4
2% (w/v) Aprotinin
0.2% (w/v) SDS
300 mM NaCl
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| Bovine Gamma Globulin Solution |
| 1 mg/ml in ddH2O
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| Elution Buffer |
| 0.1% (w/v) SDS
0.05 M Ammonium Bicarbonate
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Sodium Chloride
Ammonium Bicarbonate
SDS
Aprotinin
Trichloroacetic Acid
Tris
NP-40
Ethanol
2-Mercaptoethanol
EDTA
Sodium Deoxycholate
Bovine Gamma Globulin Solution
Elution Buffer
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1. If the polyacrylamide gel requires the use of radioactivity to detect the protein, observe the rules and cautions when using radioactivity.
2. The bands may be excised from either dry or wet polyacrylamide gels. If excised from a dry gel, simply remove the paper backing before placing in the Elution Buffer.
3. Corex tubes work well.
4. If a large amount of acrylamide is involved, more than 3 ml of Elution Buffer may be used.
5. The bovine gamma globulin is a carrier protein. BSA may also work as well or better at this step, but BSA has not been optimized for this protocol.
6. If the protein was labelled radioactively, the supernatant should be disposed in the radioactive waste.
7. Split the sample into two tubes. Use the second sample as a control serum to show that the immunoprecipitation is specific.
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