Hi,

I don't know about the buffer included in that kit, but we use mostly the buffers below for a CoIP of a binary complex with Dynabeads( sheep anti mouse IgG). It works well and one of the proteins in that complex is also a tail anchored protein as well.

Binding buffer (BP)

20 mM HEPES, 110 mM KAc, 5 mM NaAc (x 3 H2O), 2 mM MgAc, 1 mM EDTA pH 7,3 adjusted with 5 M KOH

Blocking Buffer

BP+ 0.1 % BSA

Washing or Equilibration Buffer

BP+ 0.5% Triton-X100 + 1:200 Protease Inhibitor cocktail.

Best

Radioimmunoprecipitation (RIPA) buffer is commonly used for immune precipitation studies, but the exact composition varies between authors:

• 10-50 mM Tris-HCl pH 7-8
• 150 mM NaCl to keep the osmotic pressure near physiological
• nonionic detergent (1% Triton or NP40) to prevent non-specific interactions with each other or the tube
• anionic detergent (0.1-0.5% desoxycholate, 0.1-0.5% SDS). This needs to be optimised for every assay, the higher the concentration, the cleaner the result, but the lower the signal.

The following ingredients are optional and included as needed:

• Protease inhibitors (1 mM PMSF (fresh from 1 M stock in i-propanol), 1 µg/ml each leupeptin, aprotinin, pepstatin, 1-5 mM EDTA, 0.5-1 mM EGTA, 5 mM aminocaproic acid).
• 1 mM each Na3VO4 and Na4P2O7 as phosphatase inhibitor (if phosphorylation status is important)
• 1 mM NaF as preservative
• 5-10 mM DTT or ßME as antioxydant. DTT is more expensive than ßME, but its redox potential is better suited for the Cys-Cys bond.

since you are immunoprecipitating a membrane protein, you will need detergent in your binding buffer. 1% Triton X-100 or NP-40 is commonly used. 0.5% SDS is probably too high for co-IP.

typically you want a buffer (Tris or HEPES pH 7.5 is common)

salt (e.g. 150 mM NaCl, though depending on the specific application there are other options)

consider whether divalent cations (Mg, Ca) are necessary for your protein-protein interaction

protease inhibitors

possibly phosphatase inhibitors

Hi,

The co-IP kit was developed to mimic the intracellular conditions while providing customized stringency for different protein complexes in a cell solution. It offers the opportunity to optimize and fine tune weak and transient interactions between short amino acid sequences in different proteins.

The kit consists of 2 components, 1 - beads and buffers for Ab conjugation and 2 - buffer system for easy fine tuning and optimization for each protein - protein interaction (ref Albert et al. 2007 (Nature 450, 683-694))

Buffer modifiers

• Sodium Chloride (e.g., 1 M NaCl)

• Dithiothreitol (e.g., 1 M DTT)

• Magnesium Chloride (e.g., 1 M MgCl2)

• Potassium Acetate (e.g., 1 M KOAc)

• Tween™-20 • Triton X-100

• Protease inhibitors without EDTA

The buffers are only sold as part of the co-IP kit.

Feel free to get in touch at [email protected]

kind regards

ketil