Back

Cleavage Site-Specific Antibodies

As different proteins are processed in many different ways, Cleavage Site-Specific Antibodies (CSSAs) can also be very useful tools for your research. First, it is important to start with a quality peptide. In our packages for pan antibodies, a purity of >85% works just fine, but we have found that >90% purity works best for CSSAs. In order to get the most specific antibody possible, we prefer to make two separate peptides: a peptide for the exact cleavage site and a peptide that spans this region. We use this spanning peptide as a negative control and to deplete antibodies that react with the full-length protein from the serum.

After the sera is generated, we will discuss the resultant ELISA with you before proceeding with affinity purification. If the results are not clear enough to indicate how to proceed, we can send you samples for testing in your system. Since the specificity of a response can vary greatly between rabbits, it is important to use the best material for the purification.

Once the material to be used is decided upon, we have a method for making sure you receive the best antibody possible from your project. The double affinity purification mentioned below means that we first incubate the sera with an affinity column using the spanning peptide to deplete any non-specific antibodies that are available. The flow-through from this is then incubated with an affinity column using the cleavage peptide. The cleavage-site specific antibody is then eluted off this column and sent to you.

Keep in mind that every project is different and the relative responses just as varied. We have seen and done enough, however, to be able to accommodate these variances with adjustments to the protocol. Have no fear, the expertise of NEP is here to help!

CSSA Rabbit Package: $3,250 (90 days)

• Antigen design/Bioinformatics

• Two peptides, 14mg CSS-peptide, 10mg spanning peptide at >90%

• 5mg uncoupled to customer, 1mg for ELISA, 4mg affinity column each peptide

• 4mg CSS-peptide coupled to KLH

• Mass Spec and HPLC trace (each peptide)

• Three immunizations of two rabbits (New Zealand White)

• 5mL pre-immune serum per rabbit

• Two production bleeds (40-50mL antiserum) per rabbit

• ELISA on 1^st production bleed (each peptide)

• Double affinity purification of 40-50mL antiserum to yield cleavage site-specific antibody

Immunization and Bleed Schedule:

Day 0 Pre-immune

Day 0 Boost (CFA)

Day 14 Boost (IFA)

Day 28 Boost (IFA)

Day 35 Production Bleed #1

Day 42 Production Bleed #2

Please note this is described as a rabbit package, but is available in other species as well.