I have received my sera and they are red in color. What is this and do I need to be concerned?

The red color is referred to as hemolysis and is due to the lysis of red blood cells and release of hemoglobin into the antiserum during collection and processing. This occurs intermittently among rabbit populations and is typically encountered among rabbits that are difficult to bleed. There is no reason to be concerned about the quality of serum that contains hemolysis. The presence of hemoglobin in an antiserum does not affect the performance characteristics of the antiserum.  Furthermore, any red blood cell debris that is present in the antiserum is removed during a standard centrifugation step during processing.
If desired, the hemoglobin can be removed from the antiserum by adding 45% (NH₄)₂SO₄ (final w/v) to the serum and incubated with stirring at 4°C for 2 hours.  THe material should be centrifuged and the supernatent removed.  The pellet can be re-dissolved in an equal volume of water and dialyzed against PBS (0.05% azide).

I am not seeing anything on my Western Blot. What do you recommend?

This could be due to several problems:

1. Make sure that the protein transferred properly to the membrane.  Ponceau S solution (Sigma #P7170), is a good reversible stain for checking proteins on the membrane.  Stain the membrane in this solution for 10 minutes.  Proteins will show up as pink-red on a lighter pink background.  Destain in water until the color is gone and proceed with blotting.
2. Increase the concentration of the primary antibody.
3. Increase the concentration of the secondary antibody.

I am seeing a lot of bands (high background) on my western blot. What do you recommend?

There are several things that could be contributing to the problem in this situation. Here are some suggestions:

1. Change the concentration of the primary antibody. Often times there will be high background if the concentration is too high.
2. Change the concentration of the secondary antibody. Many secondary antibodies will bind nonspecifically if they are used at too high of a concentration. Try lowering the concentration.
3. Affinity purify the serum.  This will often remove nonspecific antibodies that cause the extra bands.
4. Block the membrane for a longer period of time.  Most blocking solutions contain 2-5% dry milk.  Block the membrane in this solution overnight at 40°C (the lower temperature will keep the milk from going sour).  This will not remove high levels of background, but can eliminate lower background and make a fairly good blot look better.
5. If you are using an antibody that is specific to a phosphorylated epitope, DO NOT use milk to block.  Kinases in the milk will nonspecifically phosphorylate proteins in your sample.  Use BSA as a blocking agent.  It is important to block at least overnight.  BSA is not as good of a blocking agent and you will see more background.

My custom antiserum does not work in immunocytochemistry. What do you recommend?

This phenomenon is not uncommon and many antigens require some form of antigen retrieval, particularly proteins that form multimeric complexes, located in the nucleus or the extracellular matrix, or are heavily glycosylated.

Methods and reagents for microwave or proteolytic antigen retrieval are available from
Zymed  1-800-874-4494
or
Biogenex  1-800-421-4149

I need to attach a peptide to a chromatography resin for affinity purification of antisera or proteins. What activated affinity resin should I use?

We typically recommend conjugating a peptide to an affinity matrix through NH₂ linkages.  These are typically the most stable and can be attached either through NHS or CNBr activated resins (Pharmacia Biotech).

What should I do to terminate the project?

To terminate the project please contact our office in Houston at 1 800 234 5DNA (5362).