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