Can you use RIPA buffer for co-IP?

Yes the RIPA buffer is your best friend for co-immunoprecipitation.

How much protein do you need for co IP?

Protein extract should not be too dilute to avoid loss of protein and to minimize the sample volume to be loaded onto gels. The minimum concentration is 0.1 mg/mL; optimal concentration is 1–5 mg/mL.

Can you add a denaturant to the sample when you do co IP?

All Answers (2) Yes, that will work pretty much fine.

Is RIPA buffer compatible with ELISA?

RIPA buffer is not compatible with assays that quantify enzyme activity as the SDS interferes with the protein activity.

What is the difference between the wash buffer and the elution buffer?

The Wash Buffer is a buffered solution of 10 mM imidazole, optimized to minimize non-specific binding of proteins during the protein purification process. The Elution Buffer is a buffered solution of 250 mM imidazole, optimized to elute the bound histidine-tagged target protein(s).

How is co-IP done?

Steps in a standard Co-IP protocol.

1. Lyse your Cells. Here you gently break open your cells to make your protein accessible to the antibody.
2. Add Your Antibody.
3. Add the Protein A/G Beads.
4. Incubate.
5. Collect.
6. Wash the Beads.
7. Elute your Protein(s)
8. Detect your Protein(s)

How do you lyse cells for Elisa?

Tissue Lysates – Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris.

Does RIPA buffer interfere with Elisa?

There are many RIPA recipes around. Often they contain about 0,1% SDS and/or 1% Sodium Deoxycholate, both of which are ionic detergents which tend to negatively affect binding to the ELISA plate.