I have copied the protocols for IP and WB below. Please note these are a guideline only and may require some individual optimization.
I am happy to answer any further questions you have.
Immunoprecipitation (IP) lysis buffer
Primary Antibodies made in Rabbit
Normal IgG, negative control
Protein A Sepharose Beads
IP lysis Buffer
12.5 ml 1M NaCl (250mM)
2.5 ml 1M Tris (50mM)
500ul 0.5M EDTA (5mM)
2.5ml 10% NP-40
32 ml dH20
Protein A Beads
Resuspend 400 mg of Protein A beads in 10 ml of distilled H2O. Mix well to resuspend. Spin at 250 rpm for 5 minutes. Wash 3X in 10 ml IP Lysis buffer. Resuspend to 10 ml with IP lysis buffer for a 20% solution. Use 100 mcl per IP reaction.
4X Sample Buffer
Glycerol 4.0 g
Tris Base 0.68 g
Tris HCL 0.67 g
LDS 0.80 g
EDTA 6 mg
Brilliant Blue G250 2.5 mg
Phenol red 2.5 mg
Adjust volume to 10 ml with ultra pure water.
Store at 4oC.
1X Sample Buffer
4X sample buffer 150 mcl
1M DTT 60 mcl
Distilled water 390 mcl
Make fresh for each use.
1. Prepare cell lysate according to protocol. Place 500 mcl of the prepared cell lysate (1-3 mg/ml) into a 1.5 ml micro-centrifuge tube.
2. To this tube add 2 to 10 mcg of the primary antibody (If using neat sera or an IgG fraction such as Protein-A purified antibody, larger amounts are likely to be required. For best results, optimal amounts of antibody should be empirically defined.)
3. To a negative control reaction, add an equivalent amount of normal rabbit IgG.
4. Add 100 mcl of a 20% Protein A suspension.(Amersham Biosciences, Cat# 17-0780-01) to the mixture of antibody and cell lysate. Rotate the immunoprecipitation reactions (end-to-end) for 3 hours at room temperature or overnight at 4 C.
5. Centrifuge (200 x g; 5 minutes) to pellet the complex.
6. Remove the supernatant and add 500 mcl cold cell lysis buffer. Centrifuge (200 x g; 5 minutes).
7. Repeat wash step 6 twice more. After each centrifugation remove as much of the supernatant as possible.
8. After removing the supernatant from the third wash, add 40 mcl of freshly prepared 1X sample buffer to each tube and heat at 90 C for 5 minutes.
9. Continue with electrophoresis and immunoblotting as described under western blotting protocol. Load 8 to 16 mcl (20 to 40% of the IP reaction) to a polyacrylamide gel.
Note: For optimal results, complete reduction of the sample is required. We recommend the use of 0.1 M DTT in SDS-PAGE sample buffer and immediately heating samples, loading and running gels.
Western Blot Protocol
20X Running Buffer
Tricine (free base) 71.7 g
Tris (free base) 72.6 g
SDS 10.0 g
Sodium Bisulfite 2.5 g
Adjust to 500 ml with ultra pure water.
Store at 4oC. For 1X Running Buffer, add 10 ml of 20X Running Buffer to 190 ml of distilled water.
10X Transfer Buffer
Tris (free base) 15.2 g
Glycine 72.1 g
SDS 5.0 g
Ultra pure water to 500 ml
Store at 4oC.
1X Transfer Buffer
10X Transfer Buffer 50 ml
Methanol 100 ml
Distilled water 350 ml
Make fresh for each use.
5% non-fat dry milk in TBSTCarnation non-fat dry milk 50 g
TBST 1 liter
TBST (Tris Buffered Saline with Tween 20, pH8.0)
Tris 6.1 g
NaC l 8.68 g
Tween-20 500 mcl
Adjust the volume to 1 liter with distilled water. Adjust pH to 8.0 with HCL.
Store at 4-25oC.
Electrophoresis in SDS-Polyacrylamide Gel and Transfer to Nitrocellulose:
Many of the reagents for these procedures are commercially available. Sources that are preferred by Bethyl Laboratories, Inc. are:
Polyacrylamide gels (4-12% Tricine), running buffer and transfer buffer from PAGEgel
1. Cut open the package that contains the gel cassette and drain away the buffer.
2. Rinse the wells with distilled water.
3. Rinse the wells with fresh 1x running buffer.
4. Place the gels on the buffer core so that the shorter plates face inward. If only using one gel, use a buffer dam to seal the other side.
5. Fill the inner core with fresh 1X running buffer. If there are no leaks, fill the outer core with running buffer. Load samples.
6. Run the gels at 150V until the dye front reaches the bottom of the gel (approximately 60 minutes).
7. Soak nitrocellulose membrane and blotting paper in 1X transfer buffer for at least 5 minutes prior to opening gel cassette.
8. Open gel cassettes and place the gel on the nitrocellulose membrane sandwiched between two pieces of blotting paper.
9. Place in transfer apparatus and fill with fresh 1X transfer buffer.
10. Run transfer apparatus for 60-75 minutes on 35V.
1. Remove the membrane from the transfer apparatus and place in 20 ml of 5% non-fat dry milk in TBST for one hour, with gentle shaking.
2. Dilute the primary antibody in 15 ml of 5% non-fat dry milk in TBST. For best results, the optimal dilution of antibody should be empirically defined.
3. Incubate the membrane in diluted primary antibody for two hours to overnight with gentle shaking at room temperature.
4. Wash the membrane three times, 10 minutes each time in TBST.
5. Dilute the secondary HRP conjugated antibody in 15 ml of reconstituted 5% non-fat dry milk in TBST. For best results, the optimal concentration of the secondary HRP conjugated antibody should be empirically defined.
6. Incubate the membrane in diluted anti-Rabbit IgG-HRP Conjugate or anti-Goat IgG-HRP Conjugate for 60 minutes.
Wash as directed in step 4.
7. Develop blots with substrate solution and place in plastic membrane protector.
8. Expose membrane to film or CCD camera.