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FAQ for Custom Monoclonal Antibody Production
FAQ for Recombinant Protein Production

Custom Monoclonal Antibody Production
1. How long will it take to receive the antibodies?
2. What positive clones will I receive?
3. Do I receive the clonal hybridoma cell lines?
4. What kind of antigens are employed to make antibodies?
5. How much antigen is required for antibody development?
6. How do you test antibodies that you have developed?
7. How do you measure the antibody titer?
8. How should I store my antibodies?
9. What is the difference between the titer and the working dilution of an antibody?
10. How do I determine antibody concentration using the absorbance at 280nm?
11. What can I use to block non-specific binding in immunological techniques?
12. What is meant by monospecific reactivity?
13. What is the difference between a linear epitope and a conformational epitope?
14. Why does one see a very weak or no signal in Western blot?
15. Too many bands on a Western blot?
16. Why do I observe diffuse or moderate to intense staining of several tissue elements by IHC?

Recombinant Protein Production
1. How many custom proteins have you produced so far? What is the typical success rate?
2. What is guaranteed provided at the end of production? Do we have to pay even if there was no measurable protein produced or if the protein is still very dirty after your affinity purification?
3. Is payment due upon delivery, or before start of the project, or subject to milestones (e.g. 30% upfront, 30% upon proofen production, 30% upon delivery of purified protein)?
4. Are purification conditions native or denatured (we prefere native)?
5. Do you offer a discount for His and GST tagged versions of the same protein (normally 2 x 1800 $)?
6. Will the final protein product have biological activitiy?

1. How long will it take to receive the antibodies?
It typically takes approximately three months to receive the first batch of antibodies in supernatant format (secreted antibodies in conditioned media), delivered in 1 mL to 2 mL aliquots, which will be chosen based on screening protocols using the immunogen and ELISA. Supernatants will be used by the customer to validate antibodies and to select hybridoma populations for limited dilution and expansion of clonal populations. Once hybridoma clones are ready for shipping, a customer may then decide whether to produce antibodies from clones in their own laboratory, or Promab may proceed with large-scale production in culture or via ascites.

2. What positive clones will I receive?
It is impossible to test an antibody's application in every case, but we guarantee at least 10 positive clones against the customer-provided antigen by ELISA. If a customer requires additional screening assays to be performed, in order to validate an antibody for a specific application, such as Western., IHC, or flow cytometry, please contact customer service.

3. Do I receive the clonal hybridoma cell lines?
At the end of each project, we will ship subcloned, clonal cell lines to you either frozen, or as a live culture. Frozen stocks (2-3 vials unless more are requested) of cell lines will be stored in liquid nitrogen for up to 6 months at no additional charge. If a customer requires cell lines to be stored for longer than 6 months, nominal additional charges will apply.

4. What kind of antigens are employed to make antibodies?
Promab's immunization protocols can be adapted for native proteins, recombinant proteins, peptides, and conjugated small molecules. If the synthetic peptide is smaller than 8 kDa, it will require conjugation to the carrier molecule KLH through the thiol group of an added cysteine residue. In general, peptide antigens have a lower success rate in eliciting a strong immune response than do the larger recombinant proteins or protein fragments. Peptides, however, do have the advantage that they can be carefully designed to avoid sequence regions that are similar or identical to other members of the same protein family, thus making them potentially more specific.

5. How much antigen is required for antibody development?
If the antigen is a peptide, 5 mg of free peptide and an additional 5 mg of conjugated peptide will generally be sufficient. Less may be needed if your peptide is highly purified. If the antigen is a protein, 3-5 mgs at a concentration of 0.5-1 mg/mL is generally sufficient. Be sure to let us know which buffer was used and the protein concentration. For affinity purification of an antibody, 5 mg of soluble protein is required to prepare the immunoaffinity column.

6. How do you test antibodies that you have developed?
Antibodies are tested by ELISA and western blot, and/or IHC and/or ICC depending on the antibody application requirements of a customer. All Promab antibodies have been extensively tested. We also test antibodies using less conventional assays, including immunoprecipitation and flow cytometry, upon customer request.

7. How do you measure the antibody titer?
Antibody titer is determined by ELISA. In this method, we coat 96-well plates with antigen and wash repeatedly to remove unbound antigen. Serial dilutions of antibody are added to the antigen-bound wells and washed repeatedly to remove unbound antibodies. A labeled secondary antibody is then added and the amount of bound primary antibodies is determined by a colorimetric assay.

8. How should I store my antibodies?
Storage of lyophilized antibodies: All of our products are shipped lyophilized (freeze-dried). In this form they are stable without loss of quality at ambient temperatures for several weeks to months. They can be stored at 40˚C for several years. After reconstitution by addition of water, storage conditions depend on the type of antibody, as described below.

Ascites fluid: When ascites fluid is reconstituted, small amounts of azide or thimerosal are added to prevent microbial growth. Ascites fluid should be stored frozen. Monoclonals usually do not lose an appreciable amount of activity from freeze-thawing, but aliquoting is recommended to avoid repetitive freeze-thaw cycles. Prolonged storage at 4˚C is not recommended! Unlike serum, ascites fluid may contain proteases that will ultimately degrade the antibodies. Addition of protease inhibitors may help to slow degradation.

Purified IgG: Do not store dilute (<0.1mg/ml) antibody solutions unless carrier proteins such as BSA are added. IgG, like other proteins, binds non-specifically to glass and plastic. Any IgG solution below 0.1 mg/ml protein will adhere to the storage vessel and denature, thus losing activity. Repetitive freeze-thawing of dilute purified IgG is almost certain to lead to substantial loss.

Rabbit serum: Antibodies in serum are more robust than in ascites fluid. With anti-microbial additives, they may be stored at 4˚C (serum does not contain significant protease activity; in fact, serum itself contains a powerful cocktail of protease inhibitors). Frozen storage, however, is preferable for longer periods (months to years).

Polyclonal affinity-purified antibodies: Purified antibodies are less robust than in sera, since protease inhibitors and carrier proteins are removed during purification. Hence, storage at 4˚C for prolonged periods (i.e. several weeks), is not recommended.

9. What is the difference between the titer and the working dilution of an antibody?
The titer of an antibody refers to the lowest concentration (highest dilution) at which the antibody is still effective in a given system. The working dilution is usually the concentration that provides good sensitivity with a high signal : noise ratio. Using too high a concentration is wasteful and may give rise to non-specific signals. On the other hand, using too low a concentration will lead to a loss in sensitivity.

10. How do I determine antibody concentration using the absorbance at 280nm?
The amount of antibody can be determined by absorbance at 280nm. 1 OD = approximately 0.75 mg/ml of purified antibody.

11. What can I use to block non-specific binding in immunological techniques?
This will largely depend on the type of antibody used. For most ELISA and Western applications, 2% non-fat milk is an excellent blocking agent. Alternatively, 3% bovine serum albumin is sometimes as effective. Gelatin or serum (other than the species of primary antibody) may also be used to block non-specific binding.

12. What is meant by monospecific reactivity?
A monospecific antibody is one that reacts with only a single antigenic determinant. Monoclonal antibodies are the only truly monospecific reagents. The term is also used, however, to describe polyclonal antibodies that were raised and affinity-purified against a peptide antigen.

13. What is the difference between a linear epitope and a conformational epitope?
A linear epitope consists of about 6 to 10 adjacent amino acids on a protein molecule that is recognized by an antibody. In contrast, a conformational epitope consists of amino acids that are not arranged sequentially. Here the antibody recognizes only the 3-dimensional structure of the antigen. When a protein molecule folds into a 3-dimensional structure, the amino acids forming the epitope are juxtaposed, enabling the antibody to recognize the sequence.

Knowledge of the differences between linear or conformational epitopes is of significant importance in immunological applications. In a denatured protein, only the linear epitope may be recognized. Hence in protocols where a denatured protein is used, such as in Western blotting, an antibody that recognizes a linear epitope is preferred. Sometimes an epitope is inside the protein relative to its native conformation. The epitope is then inaccessible to the antibody in a non-denaturing protocol, such as a non-denaturing immunoprecipitation. A conformational epitope, by definition, must lie on the outside of the folded protein. An antibody that recognizes the conformational epitope is suitable for mild, non-denaturing procedures, such as non-denaturing immunoprecipitations or flow cytometry.

Optimally, an antibody that recognizes a linear epitope on the surface of a normally folded protein will work well in both non-denaturing and denaturing protocols.

14. Why does one see a very weak or no signal in Western blot?
There are several possible causes for this. In some cases, the antibody may recognize a conformational epitope that cannot be detected under natural conditions. ELISA, immunoprecipitation, or another appropriate immunochemical assays can be used to confirm antibody integrity. In other cases the antibody may have low affinity. Here one can increase the incubation period and the concentration of primary antibody. Weak signal can also result from an inefficient transfer of antigen from the gel to the membrane. This is caused by insufficient contact, due to the presence of air bubbles between the gel and the membrane, or due to an excess of SDS in the gel or buffer.

15. Too many bands on a Western blot?
It is not uncommon that, contrary to the theoretical predictions, several bands are detected. Although it is possible that the antibody is not entirely specific for the protein, other factors may be responsible:

a. Proteolytic breakdown of the antigen. This is not uncommon, particularly if samples are stored for prolonged time or if proteins or membranes are fractionated after homogenization of the starting tissue. All additional bands are of lower apparent molecular mass than the full-length protein. Particularly susceptible are synapsins and synaptotagmins. Addition of protease inhibitors such as PMSF, pepstatin or leupeptin should be considered. Several vendors offer protease cocktail reagents for this purpose. It is possible, of course, that some of the protein was degraded prior to extraction, in which case it is not possible to remove these products.

b. Insufficient denaturation and/or reduction. If the sample buffer does not contain sufficient SDS and/or reducing agent (DTT, 2-mercaptoethanol), the protein may not be fully dissociated into its subunits, reduced or denatured. Hence, extra bands may appear above the desired protein on the gel. Boiling the sample in sample buffer immediately prior to loading can reduce these noncovalent interactions or disulfide linkages. Use of an irreversible reducing agent such as TCEP, instead of DTT or beta-mercaptoethanol, may also be helpful.

c. Too much protein per lane or detection system too sensitive. Overloading of the gel is one of the most common reasons for "ghost bands". Immobilized proteins may provide a concentrated adsorptive surface to which certain IgG may bind nonspecifically. Similarly, such nonspecific binding may be uncovered when highly sensitive detection systems such as enhanced chemiluminescence are employed. A dilution series of the starting material usually clarifies which of the signals are artefactual.

d. Ineffecient blocking. A variety of different blocking agents are described in the literature including nonionic detergents and/or proteins. Change of the blocking conditions may remedy the problem.

e. Concentration of antigen too low. The resolution of SDS-PAGE is limited to 50-100 bands. If the relative concentration of the antigen of interest is too low (less than 0.2% of total protein), it may be difficult to detect (for instance, synaptobrevin/VAMP comigrates with histones in cell homogenates which interfere with its detection). Signal enhancement may then lead to the appearance of artifactual bands. Enrichment of the antigen by fractionation or by immunoprecipitation should be considered.

f. Unwanted specific reactivities. Antibodies, especially polyclonals, will sometimes recognize proteins in a biological sample with similar sequences as the antigen, such as those in the same protein family. Non-affinity-purified polyclonal antibodies will often recognize other bands due to the exposure of the animal to various other proteins in its lifetime. This can be addressed by requesting pre-immune serum and using this as a comparison.

16. Why do I observe diffuse or moderate to intense staining of several tissue elements by IHC?
Diffuse staining occurs when the specimen is fixed for too long, resulting in masking of antigenic determinants due to aldehyde cross-linking and increased hydrophobicity of tissue. Use of trypsin or other proteolytic enzymes will breakdown the cross-linking and render some tissue antigens reactive. In other instances, the sections may be too thick or the specimens may contain crushed or necrotic elements. This leads to a false negative staining accompanied by intense background staining. On the other hand, using an insufficiently diluted primary antibody leads to an intense staining of specimen or positive control.
1. How many custom proteins have you produced so far? What is the typical success rate?
We have made more than 150 custom proteins with a success rate of approximately 75% (yield at 4mg or more from 2 liter culture and purity at 75%).

2. What is guaranteed provided at the end of production? Do we have to pay even if there was no measurable protein produced or if the protein is still very dirty after your affinity purification?
There are two options in terms of protein production services:
Option 1): 3-5mg purified protein with 75% purity will be GUARANTEED. Otherwise no payment will be requested. The charge will be $3,100 for cloning, protein expression services, and purification in E.coli.
Option 2): You will get whatever amount of protein purified from 2 liters of culture with our best-optimized procedure. But NEITHER YIELD NOR PURITY WILL BE GUARANTEED. The charge will be $1,800 for cloning, protein expression services, and purification.

3. Is payment due upon delivery, or before start of the project, or subject to milestones (e.g. 30% upfront, 30% upon proven production, 30% upon delivery of purified protein)?
We charge subject to milestone with 50% upfront and 50% upon the completion of the project.

4. Are purification conditions native or denatured (we prefere native)?
Most of them are denatured, although native is not excluded. If you select option 2), we could try the native condition with out extra charge.

5. Do you offer a discount for His and GST tagged versions of the same protein (normally 2 x 1800 $)?
If you already have expression vector with His or GST and no new construction is needed, you will get $600 off from the original price. But we prefer to make new construction since we have more confident in using our own parent vectors for protein yield.

6. Will the final protein product have biological activitiy?
In most of cases, the protein will be purified from inclusion body and the activity has not been tested although the soluble form can be obtained sometimes. We could provide refolding service for with extra charge. Talk to our customer support for detail.