assay protocol

Troubleshooting of IP:

What if I do not get the target protein I want? Or the final protein I get is too much little? It's possibly due to the following:

• The concentration of target protein is too low or no target protein in sample

Change the sample or overexpress target gene in cell line.

• The protein A/G-agarose beads added are not enough, or they do not recognize primary antibody

Increase the protein A/G-agarose beads to 60μl/mg total protein;

Check the affinity of protein A/G-beads and IgG

• The reaction time of antibody-protein A/G-agarose beads is too short

Follow the protocol and that time should not be less than 2 hours.

• Target protein degrades

It's better to use fresh sample to perform IP experiment. Make sure to keep your sample on ice or at 4°C when IP is processed.

• The non-relevant proteins are too much

Centrifuge your sample at 100,000g for 30 min before adding antibody and protein A/G-beads to eliminate membrane fragments and insoluble proteins.

• The target protein doesn't dissolve or doesn't dissolve sufficiently

Optimize the recipes of your lysis buffer based on properties of your target protein.

• Antibody is not enough

Increase the concentration of specific antibody. Make sure antibody is purchased from a trustable manufacturer.

• The antibody's affinity is weak

Change to a high affinity antibody.

• The antigenic determinant supposed to be recognized by antibodies is sheltered by other interacted proteins

Change the antibody.

• The antibody doesn't recognize the natural conformation of target protein

Change the antibody.

• The washing conditions are severe

Reduce times of washing, or decrease salt concentration, or change to a more mild detergent

• Not enough sensitivity for downstream experiment

Check out the western blot reagents or mass spectrometry

What if there is severe non-specific binding or cross-reactivity? It's probably due to the following reasons:

• Protein A/G-beads non-specificly bind other proteins or cross-react with other proteins

Treat cells with protein A/G-beads prior to the adding of antibody, or block protein A/G-beads with 2% BSA

• Not a good specificity for the antibody

This can be confirmed by the negative control (IgG or non-relevant antibody). Try another antibody.

• The detergent is too mild

Optimize your lysis buffer recipe with a stronger detergent or increase its dose.

• The washing condition is too mild

Use a high salt concentration (>150mM NaCl) or other strong surfactants

What if bands of target protein are close to bands of heavy chain or light chain of IgG?

Try two methods:

  • 1. Make sure antibodies applied in IP and western blot are form different species.
  • 2. Use cross-linking reagent DSS to cross-link antibody and protein A/G-beads in IP.