IP Protocol

IP Protocol PDF

Immunoprecipitation

protein G sepharose preparation

Wash total beads with 1ml of 0.1% BSA/1xPBS once
↓ø 20 sec
Re-suspend in appropriate volume of 0.1% BSA/1xPBS

prot G preparation /sample
  A B C  
prot G Sepharose (50% Slurry) 30 30 30 µl
antibody A (500µg/ml) 2     µl
antibody B (1000µg/ml)   1   µl
control IgG (400µg/ml)     2.5 µl
0.1% BSA/1xPBS 1 1 1 ml

Use large bore tips
For mAbs, use 1µg initially
Sepharose can be used up to 100µl per sample
30 µl of Sepharose = 15µl bed volume
Prepare more control beads if you perform pre-clearing

 

Rotate at 4℃ 3 hrs to O.N.

ø 20sec and wash ppt with 1ml of 0.1% BSA/1xPBS
↓ø 20 sec
Wash ppt with 1ml of 0.1% Triton X-100/1xPBS
↓ø 20 sec
Repeat 2 more washes
Re-suspend in appropriate volume of 0.1% Triton X-100/1xPBS (e.g. 40µl/sample)
Keep beads on ice

sample preparation

Example of sample preparation
  XA XC YA YC  
Protein X 5 5     ml
Protein Y     5 5 ml
antigen A 3   3   ml
control w/o A   3   3 ml
10% TritonX-100 80 80 80 80 µl
total amount 8 8 8 8 ml

Add Triton X as a detergent to reduce non-specific binding (final 0.1%)

Need to duplicate if you use control IgG sepharose

Take samples of the input

Incubate on ice for 1hr

Incubation with beads

Add protein sepharose 40µl each

Rotate at 4℃ for 3 hrs to O.N.

Wash and Elution

ø at 4℃ 1000 x g for 5 min. Aspirate supernatant and transfer ppt to 1.5ml tube

Wash ppt with 1ml of 0.1% Triton X-100/1xPBS
↓ø 20 sec
Repeat 2 more times

Elute with 0.1M Glycine HCl pH2.5 40µl
↓rotate at RT for 5 min
↓ø 20 sec
Collect supernatant and mix with loading buffer

WB sample
  x1 x4
sup 40 µl - µl
0.5 M Tris (no pH)/1.5 M NaCl 3.8 µl 15.2 µl
4x LDS 15.5 µl 62.0 µl
10x SRA 6.5 µl 26.0 µl
total amount 65.8 µl 103.2 µl

Incubate at 70℃ for 30 min

Western Blotting