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Storkbio Ltd
Kämmu, Kupu, 74610 Kuusalu, Estonia
Phone +372 6604793
www.storkbio.com
info@storkbio.com
 
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Amounts of antigen needed to produce polyclonal antibodies in two rabbits
Concentrations and buffers of protein solutions used for immunization
Shipping of your antigen
Host species and number of animals
Changes in antibody production protocol
Antibody determination in serum samples
Appropriate peptide length for antibody production
Peptide selection
Peptide conjugation to carrier protein and additional Cysteine
Keeping free or blocking the terminuses of the peptide
Production of phospho-specific polyclonal antibodies
QC information provided with peptides and antibodies (antiserums)
Storage of serums and antibodies
Storage of peptides
Crude peptide vs pure peptide
M+Na and M+K mass peaks in MALDI spectra


Amounts of antigen needed to produce polyclonal antibodies in two rabbits

- Purified or recombinant proteins in solution, SDS-PAGE gel or precipitated: 500 ug - 2 mg
- Peptides and Peptide-Protein Conjugates: 2 - 5 mg

In general, its always better to provide more antigen if possible, to ensure higher antibody titer and success of your project. Also, smaller amounts of antigen can be used, but risk for low antibody production is higher. Good titer depends also upon the antigenecity of a given antigen and host species. Mammalian proteins can be less immunogenic in mammalians and because of that, animal species used for antibody productions must be well considered and also higher amounts of antigen used.

Concentrations and buffers of protein solutions used for immunization

Proteins should be provided with concentration of 1mg/ml or higher. Its better to send the protein as concentrated as possible, we will dilute the sample to suitable amounts. The buffer can be any biological buffer, eg PBS, Tris, Borate, Phosphate, without any strong denaturants and detergents. If your sample includes eg Urea and Imidazole, try to dialyze against regular buffer containing this compounds in minimal concentration (not more than 2M of Urea and 100mM of Imidazole) and try to high up concentration (eg 5 mg/ml). This compounds are harmful to the animal and must be avoided as much as possible. Also, precipitated proteins can be used for immunization without any loss in project success.
In case of SDS gel, carefully cut out the band, trying to minimize the amount of SDS and polyacrylamid.

Shipping of your antigen

You can send the sample with overnight dispatch at room temperature, with cold packs or at dry ice, depending on the storage conditions of your antigen. Storkbio cooperates with many couriers, but would like to suggest TNT and for customers in Helsinki - DPD. We can also transport the antigen with our customer numbers and add later the real costs to the final invoice, if you would like.

Host species and number of animals

The main factors in choosing species and animal numbers for immunization are antigen source and expected antisera volume. Different animals show variation in antibody titer and even in quality, so we suggest to use at least two animals, which is also our standard rabbit protocol. Using two animals lowers the risk, that whole projects turns out to be unsuccessful because of animal variation and in the same time helps to avoid usage of additional animals of same species in new project, because if titer is low in two animals, changes in species and in antigen presentation must be made. Decisiona based on results from on animal are not reliable. Also, if low antibody/antisera amount is needed, smaller animals, eg guine pigs, rats and mice should be used. For high amounts, it isn’t always better to use more than two animals. We suggest to start with two animals and in case of good titer, additional boosts can be made and decision about adding new animals to the production. It is sometimes recommended to include large number of animals only if the desired antibody is expected to distinguish closely related isoforms of the antigen (protein) within or across the species. Preferably antigen source and species should be distant and eg antigens purified from rabbits should be injected into goat, g. pig or chicken to produce high titer antibodies. Many mammalian proteins are very conserved and good titer is possible only in non-mammalian hosts such as chicken or fish. We suggest to run a simple or more complicated blast, to be aware about possible throwbacks.

Changes in antibody production protocol

We can follow a given immunization and bleed protocol if it has proven to work for a given antigen or if there are reasons to believe that Storkbio’s protocol will not give a desired response. In addition, we can utilize alternative adjuvants if supplied by the researcher. Alterations in injection and bleed protocol can also be done after the expiration of standard protocol.

Antibody determination in serum samples

Antibody testing is done by ELISA using the free peptide or protein coated ELISA plates (excl SDS-gels antigens). Antibodies to the carrier proteins are not detected this way. We will provide a complete ELISA report indicating titer of antibody in each animal.

Appropriate peptide length for antibody production

Generally we recommend to use peptides with length 11-20 amino acids. Shorter and longer peptides can fail in specificity, giving too few or too many epitopes and having potential sequence homology with proteins from same or other families. Production of peptides longer than 20 aa increases cost considerably, but in case of need we can make up to ~100 aa long peptides.

Peptide selection

Storkbio provides also sequence analysis to find suitable peptides for antibody production (no charges). Customer should send us peptide or protein sequence or gene accession number of the protein. We will analyze potential antigenecity, hydrophilicity, flexibility and surface probability of the sequence regions and in case of protein sequence, we will suggest 2-3 peptides for final selection. All recommended peptides will be compared for homology with other proteins by BLAST. It is good to provide us additional information about (possible) modifications, about regions which should be avoided or on the contrary, are of interest (N- or C-terminus, any specific domain). All peptides selected by us will be from no-Cys regions, except in case Cys remains in either of the terminuses (Cys will be used for single-point conjugation with carrier protein).

Peptide conjugation to carrier protein and additional Cysteine

All small peptides must be coupled to a carrier protein (KLH, BSA, Ovalbumin, etc) in order to elicit high titer antibodies. Generally, peptides can be coupled to other proteins by utilizing a free NH2 or COOH, but this chemistries should be avoided as most peptides contain several NH2 and COOH groups available in a given peptide sequence resulting into multi-point attachment and peptide distortion. So, we suggest to add one Cys to N-or C-terminus. Chemical conjugation using Cysteine offers a single point attachment provided there is just one Cys in the sequence (added or part of the native sequence). It is preferable to add Cys at the NH2 terminus if the peptide is internal or it represents the very C-terminus. This will keep the COOH free as it exists in native protein. For peptides representing the very NH2-terminal sequences, Cys should be added to the C-terminus of the peptide. For internal peptides, Cys can be added to either end but it is easier to synthesize peptides containing a N-terminal Cys. Cysteine can also be used to couple peptides to Sepharose for affinity purification of antibodies.

Keeping free or blocking the terminuses of the peptide

In order to mimic a protein’s physical and chemical properties, you should request peptides that have a similar structure and charge to the protein. For peptide sequences that represent the N-terminus we recommend keeping the N-terminus as NH2 like in the protein, and modifying the C-terminus with an amide group to mimic a peptide linkage. For peptide sequences that represent the C-terminus we recommend keeping the C-terminus as COOH like in the protein and modifying the N-terminus with an acetyl group to mimic a peptide linkage. For internal peptides both ends of the peptide should be modified (N-terminal acetyl and C-terminal amide) to mimic both peptide linkages. Also, peptide with both terminuses blocked, are more stable, but in case later conjugation of the peptide to any matrix should be done, its good to have amine in N-terminus.

Production of phospho-specific polyclonal antibodies

For phospho-specific antibody production two peptides are prepared – one with phosphorylated amino acid (Ser, Thr, Tyr) and the second with regular amino acid. Modified amino acid should be in the middle and peptide should be preferably ~15 amino acids long. Immunization will be done with phospho-peptide, ELISAs are later made against both peptides. Following two-step purification is first made against non-phospho-peptide and second purification against phospho-peptide. Final sample will be tested against both peptides, in case of need, on more purification will be carried out.

QC information provided with peptides and antibodies (antiserums)

All peptides are analyzed by MS and HPLC. MS and HPLC pictures will be added to Product Specification sheets. All antiserums (except in case of protein in SDS gel immunization) are ELISA tested and results will be added to Product Specifications sheets.

Storage of serums and antibodies

After receiving your serums and antibodies or by first usage, we suggest to decant the bulk (~30 ml of serum, ~2 -5 ml of antibody) into smaller amounts, if possible into experimental units and for very long term store the samples at -80°C (two years and longer), for longer (regular) term at -20°C and batches in usage, can be kept at +4°C (2-3 weeks). This helps to avoid repeated thawing and freezing, which harm your antibodies. If sodium azide is added to antiserums and antibodies (by request), you can keep the products at +4°C, but you cannot use this antibodies with living cells.

Storage of peptides

You can keep your peptide lyophilized or in solution (preferable for short time, in usage time). All peptides will be sent lyophilized at room temperature with parafilm capped to avoid moisture. Lyophilized peptides should be stored away from heat, light and moisture. Under these conditions lyophilized peptides are stable at room temperature for days to weeks, for longer term storage, peptides should be stored at least at +4°C (even at -20°C). In solution (preferable keeping at +4°C) some slow degradation reactions could take place, the rate of which will be sequence dependent. Possible degradation reactions in solution include: oxidation of Cys, Met and Trp; deamidation of Gln and Asn to Glu and Asp respectively and oxidative cyclization to form Cys-Cys.

Crude peptide vs pure peptide

Crude peptide is a mixture of desired sequence and small amounts of terminated sequences, deletion sequences and chemically modified sequences from side-reactions of either the synthesis or the cleavage step. Non-peptide impurities are TFA salts (mainly tert-butyl trifluoroacetate) and very small amount of organic solvent and scavenger residues. The main solvents used during production are NMP, DCM, DMF, piperidine and methanol. Scavengers are 1,2-ethanedithiol and triisopropylsilane. Final step before lyophilization is typically carried out in either, minimizing any non-peptide impurities and lyophilization removes volatile organic scavengers and solvents. So, we can actually say, that crude peptide includes the desired product and other sequence impurities.

HPLC purifications eliminates terminated, deletion and modified sequences, also non-peptide impurities if there are any left, giving the desired product in requested % and very small amounts of TFA salts.

M+Na and M+K mass peaks in MALDI spectra

It is very common to see Na (sodium) and K (potassium) adducts in the MALDI spectrum. The sodium and potassium comes from the water used in the peptide solvents. Even distilled and deionized water has trace amounts of sodium and potassium ions, which can never be entirely removed. These become ionized during the MALDI mass spec process and bind to the free carboxyl groups of the peptide. Because there is no water purification system that will remove every single sodium or potassium ion from water, seeing the sodium and potassium adducts at times is very common and unavoidable in MALDI mass spec. This is not an indication that the peptide is not pure, nor should it be confused with an incorrect molecular weight.

Last updated 01.03.2010

 
 
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