You can find here our frequently asked questions (FAQ) about our products and services that ProMab offers. If a question you are looking for is not here, feel free to contact our customer support.
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%).
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.
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, charging per phase. Otherwise, we ask for 50% upfront and 50% upon the completion of the project.
Are purification conditions native or denatured (we prefer native)?
Most of them are denatured, although native is not excluded. If you select option (2), we could try the native condition without extra charge.
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.
Will the final protein product have biological activity?
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.
Why is the SDS-Page blot of the protein at a higher MW than what I expected to see?
Yes, the final size of a natural protein, produced in mammalian cells under normal circumstances is at the molecular weight reported in literature. However, we are making a recombinant protein, with 2 tags, and in yeast cells. More specifically, the gene of interest is tethered behind a signal peptide gene to enhance secretion in the yeast system. This signal peptide is (usually) cleaved off from the final protein. However, sometimes it isn’t, which results in the final protein being a bit larger than expected. (Note that whether the signal peptide is cleaved off or not is not predictable, as this is a biological process performed by the cells.) The extra residues may or may not change the functions of the protein
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.
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, to validate an antibody for a specific application, such as Western., IHC, or flow cytometry, please contact customer service.
Do I receive the clonal hybridoma cell lines?
At the end of each project, we will ship sub cloned, 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.
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.
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.
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.
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.
How should I store my antibodies?
Storage of lyophilized antibodies: All our products are shipped lyophilized (freeze-dried). In this form they are stable without loss of quality at ambient temperatures for extremely long periods of time 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. Monoclonal antibodies 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.
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-to-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.
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.
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.
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.
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 significant 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.
How long will it take to receive the CAR-T cells?
We generally keep a good stock of all our off-the-shelf CAR-T cells and are ready to ship upon receipt of payment or PO. However, if we do not have the cells in stock, it will take approximately 2-3 weeks to receive the product.
What quality control is involved?
It is impossible to test an application in every case, but all CAR-T cells can be validated in many ways, for example: checked for transduction efficiency (Fab Ab by Flow cytometry), cell killing activity: IL-2, IFN-gamma assay; and real time cytotoxicity activity (RTCA).
What do I recieve from the millions of cells?
You will be provided a heterogenous mix of CD4+ and CD8+ T-cells that have been transduced with the CAR.
Can these cells be cultured and passaged similar to other suspension cultures?
The CAR-T cells are primary human T-cells that are fully activated and amplified. Because of this they will not expand in culture anymore, and cannot be expanded further. They are intended for immediate use in your assays upon thawing. Therefore, it is suggested that you have the experiment set up prior to thawing the cells.
Can I expand these cells and freeze some stock for future use?
These CAR-T cells have been transduced with a lentivirus containing the chimeric antigen receptor cassette, and are fully activated and amplified. They cannot be expanded further because of this.
I have an scFv sequence I would like to make into a CAR-T cell. Do you do custom development?
Yes we do! You can utilize our expertise and we can develop the CAR-T cells for you. Please contact customer support for more detail.
Do you sell the appropriate cell culture media and supplements for these cells?