Immunoprecipitation with Protein-A-Sepharose
All centrifugation steps: 3000rpm, 1 min.
- Use swollen Prot.-A- Sepharose (stored at 4 °C). (Weigh out 0.5g and let it swell in H2O for 1h or over night, centrifuge, and resuspend the pellet to a 1:1 slurry, add azide and store it in a 15ml tube at 4°C).
- Lyse a 6cm dish of cells in 2ml lysisbuffer.
- Add 2ml of dilutionbuffer, that is already in a 3,6ml NUNC-cryotube (or a tube of similar size) containing for example 5 µl (must be titrated) of the desired antibody (IgG).
- Incubate rotating head over tail 3-14 h at 4°C
- Wash 20-30% more than 40µl x sample number of Prot.-A.Seph. 1:1 slurry:
2x with H2O,
1x in the buffer your sample is in (example: lysis-buffer).
- Remove supernatant until you have a 1:1 slurry again.
- Add 40µl of the washed 1:1 slurry to each sample, incubate rotating head over tail at 4°C for 2h.
- Remove sup (eventually keep aliquots for investigation). Don´t try to remove all the sup, you will loose beads, but always leave around 50-100µl of sup!!
- Wash with 3ml:
2x buffer A
2x buffer B
1x BufferC
- Remove sup (don´t try to remove all the sup, you will loose beads!!)
- Boil in sample buffer (Most practically in boiling water because of the size of the tubes).
Lysisbuffer:
10mM Tris/Cl
150mM NaCl
1mM EDTA pH8
1% Triton-X-100
0, 5% Desoxycholat
Aprotinin/Leupeptin/PMSF
Dilutionbuffer:
10mM Tris/Cl
150mM NaCl
1mM EDTA pH8
1% Triton-X-100
0,5% Desoxycholat
1mg/ml BSA
0,5% lowfalt milk powder
Aprotinin/Leupeptin/PMSF
A:
10mM Tris/Cl
150mM NaCl
2mM EDTA pH8
0, 2% Triton X-100
B:
10mM Tris/Cl
500mM NaCl
2mM EDTA pH8
0, 2% Triton X-100
C:
10mM Tris/Cl
0, 1% Triton X-100
Hello Priyankar,
I would say Tanja's protocol is quite good. Just a few details:
1. Lysis: watch out using EDTA, many protein-protein interactions are established through Ca2+ or Mg2+, which in the cell are at 1.5 mM conc in average... Already a lysis disrupts local concentrations of divalent ions, sequestering them with EDTA might favour even more the loss of these interactions.
2. Choosing protein A or protein G beads: this is your first step in your IP and depends on the primary Ab you are using. Rabbit polyclonals prefer protein A but mouse monoclonals (IgGs) prefer protein G. When dealing with Abs from other species you'd better look on the web what they prefer.
3. Spins when you deal with Sepharose beads: 1500-2000 g for 1 min, possibly in a swing-out rotor, to obtain a more compact pellet on the bottom of the tube (spinning stronger might crash your beads, producing low yields and high background)
4. Washes: keep in mind that using 500 mM NaCl might be too stringent for certain co-IPs, it really depends on how well your two proteins interact. You might want to compare with and without this wash.
5. I always find it better after boling in sample buffer to transfer the super in a new tube, separating it from the beads... it degrades less quickly.
Good luck!
Are the proteins of your interest membrane proteins? Are you using cells overexpressing these proteins or tissue? I have been running Co-IPs in native tissue for a while and trying to get the best conditions possible to achieve efficient solubilization without affecting interaction of 2 membrane proteins. You might have to check using different lysis buffers, different antibodies etc....
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