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2. High Background (Specific or Nonspecific)

Several components in an IP or co-IP can cause binding of the support, which can be specific or nonspecific binding. Specific binding occurs when an immobilized molecule has a similar sequence or docking site as a sample component's target. Nonspecific binding can be due to charges, contact of hydrophobic surfaces, etc.

Following is a summary of the most common causes of nonspecific binding and ways to overcome them.

The lysis step
Try to avoid frozen cells, using fresh material whenever possible. If frozen material has to be used, use frozen lysates instead of cells.

The binding step:
Avoid reducing agents because they can cleave the antibody.
If biological fluids are used as samples (biological fluids may contain antibodies which will bind to Protein A, G or L, the sinobiologcal immunomagnetic beads Kit doesn't use Protein A, G or L, but relies on direct immobilization of the antibody to the magnetic beads)

Here are some advices for you:
1. Use a pre-clearing step, as described above in Additional Considerations, Pre-clearing.

2. Remove protein aggregates (spin at 100.000 g for 30 minutes.)

3. When using primary antibody, shorten the incubation step to 45 minutes.

4. When using Protein A Resin, shorten the incubation step to 30 minutes.

5. Block Protein A- (or Protein G-) magnetic beads see Additional Considerations, Protein A/G/L Blocking

6. Magnetic beads are especially recommended for IPs with cytoskeletal (or other filamentous) proteins because these proteins tend to easily precipitate. The magnetic beads coated with secondary antibodies have made co-IP possible when it had been previously impossible to perform these experiments successfully with conventional matrices.

The washing step:
Actin contamination

1. Add10 mM ATP to lysis and wash buffers.

2. Adjust washing stringency and steps.

3. Move toward a higher stringency buffer and increase the number of washing steps (refer to general tips above).

4. Use up to 1% Tween-20 (a nonionic detergent), up to 0.2% SDS (an anionic and therefore, charged detergent), or up to 1 M NaCl.

5. Alternating these high stringency washes with distilled water may help to reduce background or nonspecific binding.

6. Transfer the matrix to a fresh tube after the last washing step in order to avoid carry-over of contamination.

The elution step:
To recover the proteins during the elution step, typically a pH 2.5 – 3.0 buffer is used. This alternative to boiling the immunomagnetic beads in a Laemmli-buffer will prevent contamination of the sample by Protein A or G and nonimmobilized antibody fragments. Other "mild" elution conditions using detergents or reducing agents can be tested as well.

The detection step:
High background in Western blots can be a result of the massive presence of antibody fragments in the sample.
Depending on the sample source, contamination by lipids, carbohydrates or nucleic acids can generate background. This problem occurs much more often when working with tissues lysates than when working with cultured cells lysates.


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