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Immunoprecipitation is a method that enables the purification of a protein. An antibody for the protein of interest is incubated with a cell extract enabling the antibody to bind to the protein in solution. The antibody/antigen complex is then pulled out of the sample using protein A/G-coupled agarose beads. This isolates the protein of interest from the rest of the sample. The sample can then be separated by SDS-PAGE for western blot analysis.
The ideal lysis buffer will minimize protein denaturation while releasing an adequate amount of proteins from the sample. Non-ionic detergents such as NP-40 and Triton X-100 are less harsh than ionic detergents such as SDS and sodium deoxycholate. Other variables that can affect the success of immunoprecipitation include salt concentration, divalent cation concentration and pH. To optimize the variables, they should be tested within the following ranges (from Harlow and Lane, page 231):
Non-denaturing lysis buffer
Use for antigens that are detergent soluble and are recognized in native form by the antibody. Triton X-100 can be substituted for NP-40.
Store up to 6 months at 4°C. Immediately before use add protease inhibitors.
For convenience, a 10% sodium deoxycholate stock solution (5 g into 50 mL) may be prepared. It 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 cell lysis such as homogenization with a Dounce homogenizer.
PBS containing:
Store up to 6 months at 4°C. Immediately before use add protease inhibitors.
Denaturing lysis 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.
Store up to 1 week at room temperature.
Immediately before use add
Wash buffers
Store up to 6 months at 4°C. Immediately before use add protease inhibitor.
Protease inhibitors
Proteolysis, dephosphorylation and denaturation begin as soon as cells are lysed. Putting samples on ice will slow down these processes, but protease and phosphatase inhibitor cocktails are also available. If not using a cocktail, PMSF (50 μg/mL) and aprotinin (1 μg/mL) are protease inhibitors commonly used for immunoprecipitation.
Other reagents
Lysates from cell culture (non-denaturing)
Lysates from cell culture (denaturing)
Lysates from tissue
Pre-clearing the lysate can help reduce non-specific binding and reduce background. However, if the final detection of the protein is by western blotting, pre-clearing may not be necessary unless a contaminating protein is interfering with visualization of the protein of interest.
It is important to make sure that as much of the normal serum is removed as possible, as this will compete with the antibody against the antigen of interest. To check for this, a test can be done with lysis buffer instead of sample, performing all pre-clearing steps as above.
Running a gel of the resulting supernatant and staining with Coomassie will reveal if the serum Ig is being removed effectively. If serum has not been sufficiently removed, bands will be present at 50 and 25 kDa for heavy and light chains; its presence may contribute to a weak immunoprecipitation. Consider either decreasing the amount of serum or increasing the amount of beads incubated with your samples in the pre-clearing step.
There are a few different methods to immunoprecipitate proteins. The first approach (Method A) is to mix antibody with protein sample, followed by addition of Protein A/G support. This method yields high purity of protein; however, the antibodies are also co-eluted with protein of interest which sometimes creates difficulties in western blot detection.
The second approach (Method B) is to bind antibody to the Protein A/G beads and then mix with the antigen. This method gives lesser yield than the first one, but avoids the problem of co-elution of antibodies.
Method A
Immunoprecipitation with antibodies in solution:
Method B
Immunoprecipitation with antibody-agarose conjugate:
One of three methods can be used to elute the protein from the beads. SDS buffer is the harshest, which will also elute non-covalently bound antibodies and antibody fragments along with the protein of interest. On the other hand, glycine buffer gently elutes the protein with reduced amount of eluted antibody.
Glycine buffer elution
In this procedure, the complex is eluted from the beads by acidification using a buffer containing 0.1–0.2 M glycine, pH 2.0–3.0. The low pH of glycine weakens the interaction between the antibody and the beads. This method is advantageous as beads can be reused after removal of the glycine buffer. However, the eluted sample should be immediately neutralized with Tris, pH 8.0–8.5.
SDS buffer elution
The Ag-Ab complex is eluted from the beads by heating or boiling samples in loading buffer with denaturant SDS. This method is advantageous because the extraction method is highly efficient and the resulting sample is more concentrated.
Urea buffer elution
This method is advantageous for mass spectrometry because the sample can be digested by proteolytic enzymes.
Key:
+++ = Strong binding
++ = Medium binding
+ = Weak binding
- = No binding
Species immunoglobulin isotype | Protein A | Protein G |
Human IgG1 | +++ | +++ |
Human IgG2 | +++ | +++ |
Human IgG3 | - | +++ |
Human IgG4 | +++ | +++ |
Human IgM | Use anti human IgM | |
Human IgE | - | + |
Human IgA | - | + |
Mouse IgG1 | + | +++ |
Mouse IgG2a | +++ | +++ |
Mouse IgG2b | ++ | ++ |
Mouse IgG3 | + | + |
Mouse IgM | Use anti Mouse IgM | |
Rat IgG | - | + |
Rat IgG2a | - | +++ |
Rat IgG2b | - | ++ |
Rat IgG2c | + | ++ |
Chicken all isotypes | - | - |
Cow all isotypes | ++ | +++ |
Goat all isotypes | - | ++ |
Guinea pig all isotypes | +++ | ++ |
Hamster all isotypes | + | ++ |
Horse all isotypes | + | +++ |
Pig all isotypes | + | ++ |
Rabbit all isotypes | +++ | ++ |
Sheep all isotypes | - | +++ |
Protocols are provided by Abcam “AS-IS” based on experimentation in Abcam’s labs using Abcam’s reagents and products; your results from using protocols outside of these conditions may vary.