No Detection of Target Protein in Immunoprecipitation (IP)

Problem & Solution

1. Antigen degrading during immunoprecipitation

Ensure fresh protease inhibitors are added when cells or tissue is lysed.

2. No eluted target protein detected

Target protein may not be expressed in sample used or low level of target protein expression in the sample used. 
Check the expression profile of the target protein to ensure it will be expressed in the cells of your samples. If there is low level of target protein expression, increase the amount of lysate used. However, this may lead to increased non-specific binding so we suggest to pre-clear the lysate before IP procedure.

3. Insufficient antibody for capture of the target protein

Check that the recommended amount of antibody is being used. The concentration of antibody may require increasing for optimization of results.

4.Target protein has not eluted from the beads

Ensure you are using the correct elution buffer and that it is at the correct strength and pH for elution of target protein.

5. Antibody has not bound to immunoadsorbant beads

Ensure you are using the correct immunomagnetic beads for the antibody isotype used..

6. Incorrect lysis buffer used

Check the IP antibody datasheet to see whether the antibody detects denatured or native protein and ensure the correct lysis buffer is used. 

7. I seem to get low binding of my selecting antibody. What can I do to improve binding?

a. Verify binding/specificity of your antibody to your antigen, e.g., by ELISA.
b. Check the binding of your IP antibodies conjugated to the beads. If the antibodies do not captured or bound to the antigen, the immunoprecipitation experiment will not work.
c. If you have used the indirect method, try the direct method. Conversely, if you have used the direct method, try the indirect method.
d. Check the amount of beads and sample volume. With reference to the capacity of different beads proposed in the datasheet, increase the amount of beads or the concentration of your antibody during IP procedure.
e.Increase the incubation time.
f. Try another antibody.

8. I was able to immunoprecipitate my protein using Immunomagnetic beads before, but after crosslinking the antibody to the beads I get no protein bands on my gel.

The binding sites of your antibody have likely been altered by the crosslinking. When this occurs your antibody will show reduced affinity or no affinity to its target antigen.
This is always a high risk with crosslinking, and it is a problem easily avoided by choosing the Sino Biological primary antibody conjugated immunomagnetic beads.
Another consequence of crosslinking can also be increased affinity for unintended (non-specific) targets. 

9. I removed the non-cross-linked antibodies before performing the immunoprecipitation procedure, but still get bands on gel indicating that the antibody is coming off the Immunomagnetic beads.

If you are using reducing agents in the sample buffer before gel loading, try a sample buffer without reducing agents.
Reducing agents such as DTT or β-mercaptoethanol will break disulfide bridges within antibodies resulting in release of the antibody light and heavy chains.
Another option is to elute the protein by lowering the pH or by the polypeptide solution, this will show non-antibody band in gel.