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Phospho Site-Specific Antibodies

Given how important phosphorylation has been found to be in regulating many critical biological functions, Phospho-Site Specific Antibodies (PSSAs) can be very useful tools for your research. There are thousands of known phospho-sites - with more being discovered every day - and most have not been developed as products, making them among the most sought after items on the market today. Luckily, you have us available to lend a hand!

NEP has been making phospho-peptides and antibodies for years. 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 PSSAs. The method of phosphorylation is also important. Some companies make one non-phosphorylated peptide, split the synthesis in two, and ‘globally’ phosphorylate unprotected amino acids in order to save on costs. As the results from this can be somewhat circumspect, we prefer to make two separate peptides: a phospho and non-phospho. For the phospho peptide we only use pre-phosphorylated amino acids that we purchase from reputable vendors. Made thusly, there can never be questions about the integrity of the material that was used for injections.

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 non-phospho peptide to deplete any non-specific antibodies that are available. The flow-through from this is then incubated with an affinity column using the phospho peptide. The phospho-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!

PSSA Rabbit Package: $3,500 (90 days)

• Antigen design/Bioinformatics

• Two peptides, 14mg phospho peptide, 10mg non-phospho peptide at >90%

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

• 4mg phospho-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 phospho-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 all of our other species as well.