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Add 300 μl incubation buffer (Table 3) and an appropriate amount of primary antibody to the whole (or pre-cleared) lysate. Optimal antibody concentration should be determined by a previous titration experiment.
Gently rock the mixture at 4°C for 4 hours or overnight.
Set up a negative control with control IgG corresponding to the primary antibody source.
Add Protein A or G agarose slurry (50 μl) to capture the immunocomplex. Gently rock the mixture at 4°C for 4 h.
Centrifuge the mixture at 500–1000 rpm for 30 seconds at 4°C and discard the supernatant.
Wash the beads 3–4 times with 1X TBS or 1X PBS with 0.2% Tween 20 (or another detergent depending on its stringent and protein of interest).
Centrifuge and discard the supernatant. Keep about 60 μl of supernatant after the last centrifuge.
Resuspend the pellet with 20 µl 4X SDS Sample Buffer (Table 4), gently vortex for several seconds, and load on the gel.
|Table 3||Table 4|
|Incubation Buffer||4 x SDS Sample Buffer|
|2.7 mM KCL||12% SDS|
|1.5 mM KH2PO4||25% Glycerol|
|3.2 mM NA2PO42 12H2O||150mM Tris HCL (pH 7.0 1M stock)|
|137mM NaCl||0.03% Bromophenol Blue|
|5mM EDTA 2Na||20% B-mercaptoethanol|
|10mM NaF||Add ddH2O to the final volume, aliquot, and store at -20°C|
|Add ddH2O to 1000ml, adjust pH to 7.4||20% B-mercaptoethanol (or 500mM DTT replace) should be freshly added before use.|
The majority of bindings to Protein A or G work well in physiological conditions. Some bindings to Protein A or G can be enhanced by adapting the pH value (e.g., Protein G binds best to IgG at pH 5.0).
The washing step should not interfere with the desired protein bindings and should remove all unwanted protein bindings and debris. Perform washing and obtain elutions using gravitational flow through filter columns loaded in microfuge tubes.
Most commonly used buffers:
Most commonly used detergents:
Most commonly used additive:
If the IP sample is further used for Western blotting, the sample can be directly diluted in a SDS-PAGE sample buffer containing reducing agents. The most commonly used EB is glycine 0.1 M at pH 2.5–3.5 and 1% of SDS to disturb the bead-antibody-antigen interactions. If the antibody-protein binding does not dissociate, or if the protein becomes denaturated, the pH can be changed.