1. No eluted target protein detected
Antibody causes
1. The amount of antibody used is not enough to capture the target protein. 
Check whether the antibody concentration recommended by the supplier is used to ensure that there is sufficient antibody and antigen binding, and the amount of antibody used can be increased appropriately. 

2. Antigen antibodies do not bind. 
The inability of antigen antibodies to recognize each other is an important reason why the eluted target protein is not detected. The failure to bind antigen antibodies is usually caused by the following reasons
The lysis solution or buffer changes the natural conformation of the target protein, and the antibody cannot recognize the natural conformation of the target protein. Buy the antibody that has been verified by the manufacturer as much as possible. For example, partial immunoblot antibodies recognize only denatured antigens. 
Use appropriate antibodies and check that the concentration of the antibody is within the manufacturer's recommended range to ensure that the antibody is active. 
Sample and Reagent Problems
3. Cell antigen expression is low, or not expressed. 
Ensure target protein expression of the sample cells. If the target protein is low expressed, the amount of lysed sample used can be increased to ensure sufficient lysis so that the lysate contains sufficient protein sample. 

4. It is ensured that the addition of fresh protease inhibitors at the time of cell lysis prevents protein degradation. 

5. The affinity of the beads to the antibody is low, resulting in the antibody not binding to the beads. 
Experimental process issues
6. The target protein is not eluted from the magnetic beads
Make sure you use the correct elution buffer, which is at the right concentration and pH to elute the target protein. In some cases, extremely strong interactions may occur between antigen and antibody or binding protein binding, which cannot be destroyed by traditional elution buffers. 

2. High background
1. antibody non-specific binding
BSA did not pre-block all of the beads, resulting in binding of non-target proteins to the column. 
Using too much antibody results in non-specific binding. 
Too many cells or too much protein in the lysate leads to non-specific binding. 

2. Remnants of proteins insoluble in solvents
The supernatant was removed immediately after centrifugation, leaving the insoluble protein at the bottom of the centrifuge tube. 

3. Insufficient washing
Before centrifugation, put the cap on the test tube and turn it upside down several times to rinse thoroughly to reduce the interference of impurities. 

4. If an affinity purification column is used to enrich the protein of interest and high background is still present, it may be due to non-specific binding or cross-reaction. Try adding more elution steps or increasing the concentration of the elution buffer to reduce the amount of sample added to the beads. 

5. Contamination of sample
Contaminants such as bacteria, fungi, other proteins, etc. can cause high background in the experiment. Solutions include the use of sterile reagents and equipment, attention to the sterility of experimental manipulations, and appropriate washing steps to remove contaminants. 
3. Interference of target protein by antibody heavy or light chain

The molecular weight of the light chain and heavy chain of the denatured antibody IgG is about 25 and 50 kDa , which may mask the target protein bands with similar molecular weight. You can use the following methods to perform optimization experiments:

1. Change of antibody species source: Use antibodies of different species for IP and Western blotting to avoid cross-reactivity, e.g. use mouse antibodies for co-immunoprecipitation and sheep antibodies for immunoblotting. 

2. Antibodies using specified tags: add a primary antibody-specific tag and use a secondary antibody against the specific tag, e.g., using his or bio-labeled antibodies, to detect a secondary antibody against a biotinylated antibody. 

3. Distinguish according to antibody conformation: Use special secondary antibodies, such as secondary antibodies that only recognize non-reduced antibodies, but do not bind to reduced antibodies. The use of this type of secondary antibodies can effectively avoid experimental interference caused by ineffective binding. 
4. Distinguish according to the molecular weight of the antibody: if our target protein has no band near 25kd, light chain secondary antibody can be used for binding. 

4. A large number of antibodies are eluted
1. Optimize antibody concentration: Try to reduce the amount of antibody used to reduce non-specific binding. The antibody concentration was gradually reduced and the experiment was performed to see if the results improved. 

2. Optimization of the wash buffer: The elution step was optimized using a wash buffer containing the appropriate concentration. Different wash buffer components and concentrations may need to be tried to reduce non-specific binding. 

3. The use of a high intensity elution buffer resulted in the elution of many non-specific proteins along with the antigen. Gradient elution using a mild glycine buffer should be used instead. 

4. Addition of a suitable blocking agent to reduce non-specific binding. These blockers can occupy unbound sites, reducing binding of the antibody to non-specific proteins. 
5. Non-specific binding band or bands
Antibody causes
1. Antibodies or carriers may bind non-specifically to other proteins, resulting in increased background signals that affect the accuracy of the results. 
Lysates were pre-treated prior to immunoprecipitation and appropriate solutions or enzymes were added to reduce background signal. 

2. Using monoclonal antibodies
The specificity of the antibody used was not strong, and the experiment was carried out by trying to replace the antibody with a more specific one. Ensure that the antibodies used are validated, with specificity and high affinity. Validated antibodies can significantly reduce non-specific binding. 

3. Using too much antibody leads to non-specific binding. 
Reduce the amount of antibody used, generally because the antibody concentration is too high. 
Sample issues
4. The sample lysate contains too much protein, which exceeds the column or magnetic bead binding load, resulting in many impurity proteins in the eluate. 
5. Antigen samples were degraded without adding protease inhibitors during the experiment. 
6. Antigens exist isoforms Proteins contain multiple protein isoforms or splice variants that can migrate to different molecular weights. Post-translational modification causes the electrophoresis rate of some target proteins to be different from that of the unmodified protein. 

The reason for the experimental process
7. Insufficient closure
Beads are not sufficiently pre-blocked with BSA, resulting in non-specific binding of the sample to the column or debinding agents such as NP-40 are used to dissociate membrane proteins and reduce non-specific binding. 

8. full washing
Increase the number of washes or use a more stringent wash buffer to reduce the occurrence of non-specific binding and false positive results.