An immunoprecipitation, commonly referred to as an IP, commences by addition of a specific antibody to a prepared cell lysate, allowing for the formation of antigen–antibody complexes. Using this technique, users can look for the presence or absence of a protein, determine if a protein is up or downregulated, examine a protein's stability or post-translational modifications, or study how a target protein interacts with other proteins or nucleic acids.
There are four types of IP: Individual protein immunoprecipitation (IP), Protein complex immunoprecipitation (Co-IP), Chromatin immunoprecipitation (ChIP), RNP immunoprecipitation (RIP and CLIP).
█ Flow Chart:
█ Materials:
--Protein sample
--Antibody specific to target protein
--Protein A or G agarose beads
--Lysis buffer (containing protease inhibitors)
--Wash buffer (containing protease inhibitors)
--Elution buffer
█ Protocol:
1) Wash cultured cells with pre-chilled PBS for 2 times carefully
2) Add in cold RIPA lysis buffer
3) Scrap cells off to clean 1.5ml eppendorf tubes with a clean, cold scraper. Put them on a low-speed rotating shaker for 15 min at 4°C
4) Centrifuge at 14,000 g 4°C for 15min, then transfer the supernatant to new tubes immediately
5) Wash protein A/G-agarose beads for 2 times with PBS and make a 50% protein A/G agarose working solution (in PBS)
6) Add in 50% protein A/G agarose with ratio of 100μl for a 1ml sample solution. Shake on horizontal shaker for 10minat 4°C (This step aims to eliminate non-specific binding proteins)
7) Centrifuge 14,000g at 4°C for 15min, then transfer the supernatant to new tubes and discard protein A/G-agraose beads
8) Quantify total protein with BCA assay or other methods
9) Dilute the total protein to 1μg/μl with PBS to decline the concentrations of detergents. If you feel the concentration of your target protein is low, you can dilute the total protein to 10μg/μl. (if it’s high enough)
10) Add in appropriate amount of primary antibody to approximately 500μl total volume
11) Slowly shake antigen-antibody complex on rotating shaker at 4°C overnight
12) Centrifuge 14,000g for 5s, and keep the pellet and wash with pre-chilled washing buffer (or cold PBS) for 3 times (800μl each)
13) Collect the supernatant to proceed to SDS-PAGE, western-blot, or mass spectra analysis
█ Related Application Antibodies:
CATALOG |
PRODUCT NAME |
SPECIES |
SIZE |
DETAILS |
APAT10068 |
Human |
50ul, 100ul, 200ul |
||
APAT10097 |
Human |
50ul, 100ul, 200ul |
||
APAT10185 |
Human |
50ul, 100ul, 200ul |
||
APAT10215 |
Human |
50ul, 100ul, 200ul |
||
APAT10216 |
Human |
50ul, 100ul, 200ul |
||
APAT10217 |
Human |
50ul, 100ul, 200ul |
||
APAT10218 |
Human |
50ul, 100ul, 200ul |
||
APAT10259 |
Human |
50ul, 100ul, 200ul |
||
APAT10274 |
Human |
50ul, 100ul, 200ul |
||
APAT10296 |
Human |
50ul, 100ul, 200ul |
||
APAT10297 |
Human |
50ul, 100ul, 200ul |
||
APAT10330 |
Human |
50ul, 100ul, 200ul |
||
APAT10360 |
Human |
50ul, 100ul, 200ul |
||
APAT10457 |
Human |
50ul, 100ul, 200ul |