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ANTIBODIES 1) How much antigen is needed to make good titer antibodies?
2) What concentrations and buffers are compatible with proteins for immunization ?
3) What if the protein is NOT soluble in regular biological buffers and if it precipitates?
4) Production of antibodies using proteins embedded in SDS-PAGE gels?
5) What is the best method of shipping antigens to INBIOLABS?
6) What Species and animal number are appropriate for antibody production?
7) What options are available after the expiration of standard protocol?
8) Are other alternate immunization and bleeding protocols available?
9) How do I know if antibodies are made to the antigen and which animal is the best?
10) What happens if there are no antibodies or ELISA is negative? PEPTIDES
11) What is the appropriate peptide length for antibody production?
12) Can INBIOLABS help in selecting appropriate peptides from whole gene sequences?
13) Should I consider adding a Cysteine in peptides for making antibodies?
14) Is it necessary to use 95-98% pure peptides for antibody production?
15) How about coupling peptides to 2 different carriers (KLH and BSA)?
16) What should I consider when making antibodies to phosphorylated peptides or other modified peptdies (sulfated etc)
17) What quality control (QC) information do you provide?
18) How should my peptide be stored?
19) Can you explain the M+Na and M+K mass peaks in MALDI spectra?
20) What should I do with the ends of my peptides, keep them free or block them?
21) Who pays for shipment?
22) What happens if there are no antibodies or ELISA are negative?
1) How much antigen is needed to make good titer antibodies?
Amounts of antigen needed to produce good titer antibodies obviously depend upon the antigenicity of a given "antigen" and host species. Bacterial/viral antigens are very often highly immunogenic than mammalian proteins. In our experience, the antigen amounts given below will normally suffice to make good titer antibodies. These estimates are for using up to 2 animals using our std. protocols. - Purified and recombinant proteins: 500 ug - 2 mg.
- Peptide-Protein Conjugates: 2 - 5 mg.
- Proteins in SDS-PAGE gels: A band that is clearly visible upon staining (500 ug - 2 mg).
- Proteins as precipitates: 500 ug - 2 mg.
In general it is a good idea to provide as much antigen as practically possible (1-3 mg or more) to assure good antibody production. It doesn’t cost more to inject more.
2) What concentrations and buffers are compatible with proteins for immunization ?
In general, most biological buffers (PBS, Tris, Borate, Phosphate, etc) in moderate molarity are OK. It is desirable to send the protein at aprpox 1 mg/ml or higher. In general, you should ship the antigens the way they are normally kept or send them frozen in dry ice.
3) What if the protein is NOT soluble in regular biological buffers and if it precipitates?
High concentrations of chaotropic agents (8M Urea, GdnHCl, SDS) should be avoided. Often some recombinant antigens will not dissolve in regular buffers and require 4-8M urea etc for keeping these proteins in solution for some client’s applications. Try to minimize the concentrations of strong denaturants and detergents as much as possible by dialyzing in buffers containing lower concentration of these components. If all fails, then keep the concentration of protein as high as possible (5-10 mg/ml). It is also possible to inject proteins as precipitates. Just send the protein in buffer containing suspension of proteins. However, these extreme measures (SDS-PAGE, high concentration of urea etc, and precipitates) must be avoided and used only as the last option. Antibody titer may also be compromised.
4) Production of antibodies using proteins embedded in SDS-PAGE gels?
This method has been used successfully if amount of gel is kept to a minimum. Acrylamide gel, if injected in large amount, is toxic to animals. It is very important to load as much protein as possible on a PREP GEL (no lanes or wells), coomassie blue dye staining, destaining, and then cutting out the most dense portions of the protein band (One gel strip or its equivalent should be enough for the entire protocol). INBIOLABS will process your gel slices by mincing and homogenization before injections.
5) What is the best method of shipping antigens to INBIOLABS?
All antigens can be safely shipped overnight on dry ice. Some antigens such as peptide in powder forms or SDS-PAGE gels can be shipped at room temperature. Other antigen (proteins bound to Sepharose gels, precipitates, peptide/protein-conjugate solutions can be shipped overnight in cold-packs).
6) What Species and animal number are appropriate for antibody production?
Antigen source, its sequence conservation, and antisera volume are the primary factors in choosing a host species and their numbers. For example, antigens purified from rabbits should be injected into goat, g. pig or chicken to produce high titer antibodies. In general, peptide-protein conjugate can be reliably injected into rabbits (even if sequence of the peptide is the same in rabbit). Mammalian protein antigens, that are known to be poor immunogens, could produce high titered antibodies in non-mammalian hosts such as chicken or fish. Polyclonal antibodies generated in common animals will generally show a variation not only in their titer but also in their quality even if all animals are injected with the same antigen and bled at the same time. Therefore, it is best to include at least 2 or more animals per antibody protocol. It is often recommended to include large number of animals if the desired antibody is expected to distinguish closely related isoforms within or across the species or has other subtle changes (phosphorylated Vs non-phosphorylated). We generally recommend the use of at least 2 animals per protocol in Rabbit, G. Pig. Up to 5-10 animals are usually needed when antibodies are made in mouse/rat to collect sufficient amount of serum.
7) What options are available after the expiration of standrard protocol?
Animals on a given protocol can be extended as desired on a monthly basis only. Extension charges are determined by per diem housing, number of injections and bleeds. Please consult INBIOLABS for current charges in various species during optional extension period. Animals can be bled out at any time to collect additional blood. All projects can be extended as long as desired depending upon the availability of antigen or peptide-protein conjugate.
8) Are other alternate immunization and bleeding protocols available?
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 INBIOLABS’s protocol will not give a desired response. In addition, we can utilize alternative adjuvants (RIBI, Adjuvax, etc) if supplied by the researcher. Alterations in injection and bleed protocol can also be done after the expiration of standard protocol.
9) How do I know if antibodies are made to the antigen and which animal is the best? We recommend ELISA analyses on the first bleed to determine antibody titer. Most animals will respond by this time. A decision can then be made and the protocol modified or terminated. Antibody testing is done by ELISA using the free peptide or protein coated ELISA plates. Antibodies to the carrier proteins are not detected this way. INBIOLABS will provide a complete ELISA report indicating titer of antibody in each animal.
10) What happens if there are no antibodies or ELISA is negative?
INBIOLABS recommends ELISA on the first bleed. If results are negative, testing is repeated at no additional cost on next bleed. If there are no signs of antibody by ELISA and another technique (Western, IHC), it may be necessary to terminate the project or include changes in immunization protocol. Most custom antibody work is performed on best-effort basis. INBIOLABS has Scientist with over 20 years to experience in raising and using antibodies. We do everything possible that we know or what is recommended to us by the researchers. There are number of reasons when antigens fail to induce antibody response. It may be due the poor antigenicity of an antigen, conservation of peptide sequence in a given species, and poor conjugation of peptide to the carrier protein. Many of these factors are not in our control and we can not alter the nature of antigens or antibodies. In such cases, we offer to use other antigens. In case of peptides, you may elect to have another peptide made and we will make antibodies at no extra cost. In this case, we will only add the cost of making the new peptide and its conjugation.
No Scientist can predict functionality of antibodies. In many cases, antibodies may not work in all techniques. A given antibody may not work in blotting and be still useful for ELISA or IHC or IP’s or vice versa. This is particularly true for anti-peptide antibodies. In some cases high titer antibodies generated against the peptide may not recognize the full-length protein in Western or fail to immunoprecipitate the antigen or may not work in immunohistochemistry. Therefore, our warranties are limited to production of reasonable titer antibodies (at least 1:1K; average titer 10K-100K) by ELISA. In case of protein antibodies, we must be provided with sufficient antigen (500 ug-2 mg) for our warranties to be valid. When antigens are provided in gels or beads, our services will strictly be on best efforts basis as there are usually no good estimates of the amount of antigens in gels or beads.
11) What is the appropriate peptide length for antibody production?
In general we recommend, approx. 15-20 aa peptides if there are no constraints in selecting such peptides. Longer peptides (>20 aa) can be used but it increases the cost. We can make up to ~100 aa long peptides. It is generally not a good idea to chose peptides <10 aa unless there are valid reasons for it such as potential sequence homology with a related family member or other proteins. Shorter peptides (<10 aa) may present limited number of epitopes.
12) Can INBIOLABS help in selecting appropriate peptides from whole gene sequences?
INBIOLABS has analyzed thousands of sequences and assisted researchers in selecting immunogenic peptides. Whole gene sequences are analyzed by computer programs for antigenicity, hydrophilicity, accessibility, etc. to select antigenic peptides. It is very helpful to have additional information as to what regions of the protein (N or C-terminus, or a given domain) to specifically target or avoid. Any potential for crossreactivity with other closely related members of the same family should also be mentioned. A sequence alignment of closely related members is of tremendous help to select specific peptides. All recommended peptides are compared for sequence homology with other proteins by BLAST searches. A final selection is then made with the user input. It is a good idea to provide us with the gene accession number of published sequences or send us the sequence by email. All info shared with us always remains confidential. It will normally take 1-3 days to complete our analyses. There are no charges for this assistance or obligation.
13) Should I consider adding a Cysteine in peptides for making antibodies?
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, or a Cysteine group. 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 (non-conjugated) as it exists in native protein. For peptides representing the very NH2-terminal sequences, Cys should be added at the C-terminus of the peptide. For internal peptides, Cys can be added at either end but it is easier to synthesize peptides containing a NH2-terminal Cysteine. Cysteine can also be used to couple peptides to Sepharose for affinity purification of antibodies. Amino or COOH-conjugation chemistries should be avoided as most peptides contains several NH2 and COOH groups available in a given peptide sequence resulting into multi-point attachment and peptide distortion.
14) Is it necessary to use 95-98% pure peptides for antibody production?
Although, pure peptides are always better but it is not a requirement to have 90-95% pure peptides for antibody purposes. It is generally more economical to synthesize about 70-85% pure peptides than to spend lot more to purify peptides. The antibody project is not likely to fail because of the use of 70-85% pure peptide as opposed to >90% or better purity.
15) How about coupling peptides to 2 different carriers (KLH and BSA)?
For most anti-peptide antibody purpose, coupling of peptides to just one carrier protein (KLH or BSA) should suffice for immunization. Many small peptides will not coat well on ELISA plates. Coupling of peptides to another protein is done to facilitate coating of peptide on ELISA plates. INBIOLABS uses free peptide for coating and screening antibodies by ELISA. We have developed special technique to coat most peptides to ELISA plates. Therefore, we do not recommend coupling peptides to more than one protein and hence save these expenses.
16) What should I consider when making antibodies to phosphorylated peptides or other modified peptdies (sulfated etc)?
INBIOLABS has made many antibodies to phosphorylated peptides. Please ask for additional details.
17) What quality control (QC) information do you provide?
All peptides are analysed by MS to confirm the molecular weight. For peptides where a minimum purity has been requested we also run reverse-phase HPLC analyses. The results from these analyses are included on the Technical Datasheet (TDS) supplied with the peptides delivered.
18) How should my peptide be stored?
Lyophilized peptides should be stored away from heat, light and moisture. Under these conditions lyophilised peptides are stable at room temperature for days to weeks, for longer term storage, peptides should be stored under the same conditions but at -20 C.
As moisture will greatly reduce the long term stability of peptides, peptides should be allowed to equilibrate to room temperature in a desiccator before dispensing, thus avoiding exposure to moisture in the air which will condense on the peptide. Once dispensed, the tube should be gently purged with anhydrous nitrogen or argon, the container recapped, sealed with parafilm and stored at -20 C.
In solution, 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
Oxidative cyclisation to form Cys-Cys
19) Can you explain the 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.
20) What should I do with the ends of my peptides, keep them free or block them?
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.
21) Who pays for shipment? For custom packages a shipping charge is applied.
22) What happens if there are no antibodies or ELISA are negative?
No charge is applied.
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