Required Materials and Equipment
1.) Preparation of Lysates
Lysates from Cell Culture
2.) Pre-clearing the Lysates
Note: The preclearing step is incorporated into the procedure to reduce the amount of non-specific contaminants in the cell lysate
and to remove proteins with high affinity for Protein G or Protein A prior to the specific immunoprecipitation. The end result will be a
lowering of background and an improved signal-to-noise ratio. However, if the final detection of the protein is by immunoblotting,
pre-clearing may not be necessary, unless a contaminating protein is interfering with visualization of the protein of interest. Similarly,
this step may be skipped in cases where the protein of interest is abundant in the sample.
Note: Determine the protein concentration of the cell lysate (e.g. if performing a Bradford assay, dilute the cell lysate at
least 1:10 before determining the protein concentration because of the interference of the detergents in the lysis buffer
with the Coomassie-based reagent).
Non-denaturing lysis buffer
Store up to 6 months at 4º C
Optional: Immediately before use add protease inhibitors.
RIPA (RadioImmunoPrecipitation Assay) buffer
More denaturing than NP-40 or Triton X-100 lysis buffer, RIPA buffer contains the ionic detergents SDS and sodium deoxycholate as active constituents and is particularly useful for nuclear membrane disruption for nuclear extracts. RIPA buffer gives low background but can denature kinases.
Note: The 10% sodium deoxycholate stock solution (5 g into 50 ml) must be protected from light.
Detergent-free soluble protein lysis buffer
Some soluble proteins may not require use of detergents. Use this buffer with mechanical breakage of cells, e.g. repeated passage through a syringe or homogenization with a Dounce homogenizer. PBS containing:
Optional: Immediately before use add protease inhibitors
Denaturing lysis buffer/buffer for non-detergent soluble antigens
Epitopes of native proteins are not accessible to antibodies that only recognise denatured proteins. When harvesting and lysing the cells, heat the cells in denaturing lysis buffer. This method can also be used for antigens that cannot be extracted from the cell with non-ionic detergents. Use of DNase1 will aid extraction of proteins from chromatin.
Immediately before use add:
Washing the complexes can be done with RIPA, PBS or IP wash buffer. RIPA buffer is more stringent whereas PBS is less stringent.
1. The choice of lysis buffer depends upon the location of the protein (membrane, cytosolic or nuclear). Additionally, varying pH
as well as detergent, salt and divalent cation concentrations are steps that can be taken to optimize specificity of
1. Bonifacino, J. S. et al. (2001) Immunoprecipitation. Curr. Protoc. Mol. Biol. Chapter 10:Unit 10.16.