Q: How should I store the serum / antisera?
A: For short-term storage of the working antibody or of serum that has recently arrived, 2-3 weeks at 4°C is recommended. For long-term storage, we recommend storing the serum vials at -20°C, which is sufficient for several years. Storage at -80°C is fine also, but not necessary except for very long periods of time. The purified antibody should be aliquoted into working quantities and stored according to the guidelines above. The biggest threat to antibody stability is repeated freeze/thaw cycles. To avoid this, we recommend storing ready-to-use aliquots of the antibody and thawing aliquots only as they are needed. We also recommend the use of manual defrost freezers rather than frostless freezers.
Q: How much protein do I need to send?
A: We recommend sending 1.5 mg of the antigen for rabbits, 1.5 mg for rats and 1mg for mice. We recommend 0.5 mg/ml (better PBS or TRIS buffer) as a minimum protein concentration and welcome higher concentrations.
Q: How should I send my protein to Primm?
A: Please ship the antigen in a styrofoam box with ice packs (dry ice isn’t necessary) via FedEx, UPS or DHL Overnight to the address listed on our Order Form. To avoid additional shipping charges by these companies, please insert the Styrofoam box into a cardboard box or shipping pak prior to shipping. Please also include a printed copy of the Order Form with the shipment.
Q: What type of antigen does Primm use?
A: Primm normally makes polyclonal antibodies against synthetic peptides, recombinant or natural proteins or other antigen as required by the customer. In case of synthetic peptides, the peptide is previously conjugated with a carrier protein which in general is either Ovalbumin or KLH.
The synthetic peptides can be made directly at Primm laboratories or may be given by the customer. Also recombinant proteins can be made at Primm labs.
Q: Does the protein need to be soluble?
A: No, insoluble proteins can be used for immunizations.
Q: Can I generate antisera against gel strips containing my protein?
A: Yes, immunizing with gel bands works well for generating polyclonal antisera against a purified protein. If providing a gel strip, please stain with coomassie blue, de-stain, rinse and cut out the strip containing the target protein. Please avoid freezing or homogenizing the gel strip so that we can aliquot appropriate quantities of the protein for each immunization. With gel bands in particular, it is important to maintain a higher concentration of the protein as outlined above.
Q: Does Primm guarantee antibody titers?
A: Yes, we guarantee at least a 1:10,000 antibody titer against peptide sequences that we synthesize/conjugate. Titers are confirmed via ELISA tests that we run.
Q: Which preservatives can be added to the serum?
A: Sodium Azide is a preservative that prevents bacteria from growing in the serum (bacteria can produce proteases that denature any proteins in the serum, including antibodies). We typically recommend adding azide unless the serum is going to be used directly in cell culture applications. Azide can be removed by using a dialysis membrane with a molecular weight cutoff between 12,000 and 14,000.
Q: How will the serum be shipped?
A: Serum is typically shipped after each bleed is taken. The serum will be frozen, spun down and shipped via DHL overnight on ice pack (+4°C) with sodium azide ( NaNH3) or in dry ice without sodium azide.
Q: For peptide antigens, can Primm help the customer with the choice of the peptide sequence?
A: Yes. Primm will highlight the most antigenic regions of a protein thus allowing us to choose the most immunogenic peptides. This help is provided at no additional charge and it is based on a computer analysis of the protein sequence using algorithms that take into consideration the hydrophobicity and the knowledge of the tridimensional conformation of the different amino acid residues.In some cases, it may be better to generate antibodies with a mixture of 2 or 3 different peptides whose sequence is derived from the same protein sequence. Such approach may increase the chance to obtain antibodies able to recognize the intact protein
Q: Which are the specifications of the polyclonal antibodies?
A: The antibodies pass the quality control specs if they are able to recognize the antigen used for immunization at a dilution of the serum not lower than 1:1,000. As negative control, Primm collects the pre-immune serum which is used for the internal quality control and it is delivered to the customer together with the antibodies.
Q: How efficient is Primm protocol for the generation of antibodies?
A: Primm has developed an immunization protocol that is able to generate a good titer of antibodies in a reasonable time (7-8 weeks). Primm is aware that in some case longer protocols may lead to higher titers of antibodies. However, our protocol is an ideal compromise between quality and time.
Q: Is Primm ready to modify the protocol if required by the customer or if necessary for the success or improvement of the results?
A: Yes, Primm is available to start the immunization schedule with a protocol suggested by the customer. A specific quotation will be issued in this case. Also, in the event that the standard immunization scheme does not yield antibodies passing the established specifications, Primm may prolong or change the immunization protocol.
Q: How does Primm test the quality of the polyclonal antibodies?
A:Serum collected from the animals is tested by ELISA. According to Primm procedure, the antigen used for immunization is coated on a microtiter plate and allowed to react with the specific serum. The reaction is followed by labelled-secondary antibodies able to recognize the specific immunoglobulins bound to the antigen. The use of pre-immune sera allows to establish the specificity of the antibodies.
In case of antibodies generated against synthetic peptides, the free un-conjugated peptide or the conjugated (to a different carrier used for the immunization) peptide, is coated on the microtiter plate. In this way, we do not measure the immunoglobulins raised against the carrier protein used for immunization (Ovalbumin or KLH).
Q: What about the possibility that antibodies generated against synthetic peptides will be able to efficiently recognize the intact protein?
A: At a frequency of 10-15%, it may happen that antibodies generated through the use of synthetic peptides are indeed able to recognize the synthetic peptide used for immunization but they do not recognize the intact protein from which the peptide sequence was derived. This could be observed particularly in experiments of immunoprecipitation or immunohistochemistry.
Q: Is it necessary to purify the specific antibodies out of the serum?
A: For certain applications such as immunohistochemistry, it may be necessary that the specific immunoglobulins are purified by affinity chromatography carried out using a sepharose matrix where the specific antigen (peptide or protein) is bound on.
When the customer has no initial idea whether it may be better to purify, we always suggest that he decides later after having used the sera at the end of the immunization protocol.
The purification is routinely offered by Primm. We also provide the sepharose column carrying the antigen.