FAQs

Here are some of the most frequently asked questions we receive. Take a look below, maybe there already is an answer to your question. If not, please do contact us we will try to get back to you as soon as possible.

Click to jump to our Hybridoma Sequencing FAQ.

General FAQ

Where can I buy your antibodies?

Our antibodies are all made by us, and can all be bought directly from us. Use our online check-out or contact us to order. Alternatively, you can purchase our products from our local distributors.

What expression system are your antibodies manufactured in?

All our catalog antibodies are expressed in HEK293 cells using our HEXpress platform. Custom expression is typically performed in HEK293 cells but CHO expression is available on request.

What concentration are your antibodies supplied at?

All our reagent antibodies are supplied at 1.0 mg/ml unless indicated otherwise. Concentration and volume are clearly indicated on the vial. For bulk or custom orders antibodies can be provided at alternative concentrations.

What buffer are your antibodies supplied in?

All our reagent antibodies are supplied in phosphate buffered saline (PBS). Complete formulation is: 155 mM NaCl, 1.54 mM KH2PO4, 2.71 mM Na2HPO– 7H2O, pH7.2.

What is the purity of your antibodies?

All our reagents are produced in a serum-free mammalian cell line and so are free of common contaminants such as albumin or IgG. All our reagents are highly purified by affinity chromatography and have a minimum purity of >95%.

What are the endotoxin levels of your antibodies?

All our reagents have an endotoxin level of <1.0 EU/mg. Lower endotoxin levels can be provided on a custom basis.

Are your antibodies supplied with preservative and what preservative do you use?

Small quantities of antibody (e.g. 50, 100 or 200 μg) are supplied with 0.02% Proclin-300 (see below for information on Proclin-300). Bulk orders and custom orders can be supplied with or without preservative as requested. We may be able to provide antibodies in alternative preservatives upon request (e.g. sodium azide).

What is Proclin-300?

Proclin-300 is a highly effective biocide and an attractive alternative to other commonly used preservatives such as sodium azide and thimerosal. Its key advantages over sodium azide are that it is compatible with all enzymes (including HRP), it is effective in very low concentrations, it is active at a wide range of pHs and it has excellent stability. For more detailed features and benefits of Proclin-300 see here, and for general information on preservatives in IVD and the reasons to switch to Proclin-300 see here.

At what concentration/dilution should I use my antibody in a particular method?

The optimal antibody concentration is dependent on a multitude of factors, such as buffer composition, pH, temperature, incubation times and importantly the antibody itself. In order to obtain optimal results, researchers must therefore optimise their own experimental conditions. However, using standard protocols, it is possible to give good starting points for optimisation. For Western blotting or staining for flow-cytometry, a concentration of 1 μg/ml is good starting point, as is 0.1 μg/ml for ELISA , 5 μg/ml for IHC and 1-10 μg/ml for IP. Again, these are only rough guidelines and an initial optimisation of conditions by every researcher is recommended. Where possible, we make specific recommendations on our product data sheets.

Which antibody host species, isotype or subtype of should I choose for my experiments?

When staining samples and subsequent detection using secondary antibodies it is important not to use antibodies that will non-specifically bind to the sample (e.g. via Fc-receptors) whilst being distinct from other antibodies detected at the same time. We offer a range of Fc Silent™ antibodies to reduce background staining, and offer all our antibodies in up to seven species to allow detection by diverse secondary antibodies. For research antibodies mouse and rabbit antibodies are perhaps the most popular with the most widely available secondary antibodies.

For in vivo studies one also has to consider the host species as well as effector function of an antibody. Where possible, antibody and experimental host species should be matched to avoid the potential of an immunological response to the antibody sequence upon repeated administration. With regards to effector functions, there are two conceptual extremes: Depleting and neutralizing antibodies. For depleting function via ADCC and/or CDC mouse IgG2a/IgG2b, rat IgG2b or human IgG1/IgG3 are the prime candidates. For neutralizing functions mouse IgG3/IgG1, rat IgG1 or human IgG4 are preferable. Equally, Fc Silent™ variants can offer further reduction of interaction of the antibodies with Fc-gamma receptors. See here for more information on chimeric antibodies.

To discuss the specific requirements of your experiments, please contact support@absoluteantibody.com

What is the glycosylation profile of your antibodies?

We do not test the glycosylation profile of our antibodies, however there is much literature on the glycosylation of antibodies and other proteins in a variety of mammalian cell lines. Glycosylation is a critical consideration for in vivo experiments using glycoproteins as different glycoforms can have a substantial impact on function and half-life. It is well reported that there are differences in the glycosylation pattern of proteins produced in HEK293 and CHO cell lines and that different glycoforms impact ADCC (see here for a review on antibody glycosylation in pharmacology). However, to the best of our knowledge, there are no reports on substantial differences in ADCC, half-life or activity of the same antibody produced in both HEK293 and CHO cell lines. It is therefore common in the early stages of research to use both transient HEK293 and transient CHO platforms for production of antibodies before moving towards a stable cell line in the later development phases of a therapeutic antibody project.

Do you have a bacterial/yeast/insect expression system?

We do not currently have bacterial, yeast or insect cell expression systems. All our products are manufactured in mammalian cell lines (HEK293 or CHO-K1). We can produce any antibody format in these cells with high yields, quick turnaround times and competitive prices.

What is the allotype of your human IgG1?

Our standard human IgG1 is G1m17/G1m1/nG1m2 [also referred to as G1m(z)/G1m(a)/nG1m(x)] but we also have G1m17/nG1m1/nG1m2 [G1m(z)/nG1m(a)/nG1m(x)] available on request. For information on human G1m allotypes see this page. Other allotypes can be produced on a custom basis. For other allotype related questions please contact us directly at support@absoluteantibody.com

What expression yields do you get?

Antibody expression yields are highly variable as it is determined by the particular variable domain sequences of an antibody. We typically achieve yields of between 50 and 400 mg/litre of culture supernatant via transient transfection.

Do secondary antibodies bind to Fc Silent™ antibodies?

Yes. Fc Silent™ antibody formats feature a set of point mutations at the Fc-gamma receptor binding site, and therefore only alter a very small part of the Fc-domain. Most commercially available secondary antibodies are polyclonal, and therefore target a variety of epitopes on the Fc-domain of an antibody. Whilst it is theoretically conceivable that amongst the polyclonal mixture an antibody clone exists that has specificity to the Fc-gamma receptor binding site of the antibody and therefore loses affinity for the Fc Silent™ mutant, the vast majority of clones will be targeting a different epitope, enabling detection.

Hybridoma sequencing FAQ

What species of hybridomas can you sequence?

We have a large amount of experience sequencing mouse and rat hybridomas but we have also successfully sequenced hamster and rabbit hybridomas.

What approach do you use to sequence hybridomas?

We perform high throughput (NGS) hybridoma sequencing and identify functional antibody sequences from the total mRNA of an antibody-producing hybridoma.

How many cells do you require for sequencing?

We request 2 x 1 ml vials of cells ideally at a concentration of 1×106 cells/ml or higher. This provides us with plenty of cells which is useful in case additional sequencing runs are required to obtain a successful result.

How should I send cells to you?

Cells can be sent to us in 1 of 3 ways described below. Our strong preference is to receive viable frozen cells in the manner described in option 1.

  1. For each antibody being sequenced send a minimum of 2 x 1 ml vials of frozen hybridoma cells in freezing media (e.g. 90% FBS, 10% DMSO) at a concentration of 1×106 cells/ml or greater. Cells should be shipped frozen on dry ice.
  2. For each antibody being sequenced send a minimum of 4 snap frozen cell pellets in 1.5ml microcentrifuge tubes containing 1×106 cells or greater. Cells should be shipped frozen on dry ice. Please note if this method is used Absolute Antibody will not be able to grow the hybridoma.
  3. For each antibody send two sealed vials of live cells (e.g. 2 x 10ml) at ambient temperature. Cells should be at a reasonable density ready for immediate growth at Absolute Antibody.

What are aberrant chains/genes/transcripts?

Theoretically monoclonal hybridomas should consist of one heavy chain gene and one light chain gene which together make one functional antibody. However, many hybridomas have one or more non-functional antibody genes. These are known as aberrant genes or aberrant chains and in particular it is aberrant light chains that are most common. Aberrant chains often have frame shifts or stop codons in the sequence making it possible to identify that they are non-functional. They normally occur due to their presence in the myeloma partner cell that is used in the fusion. Many of the hybridomas derived from SP2/0 (mouse) and Y3 (rat) contain aberrant light chains but this is also true for many hybridomas derived from other cell lines.

When sequencing hybridomas great care must be taken to not mis-identify an aberrant gene as functional one. At Absolute Antibody we have a high-level of familiarity with antibody sequences and have developed a number of techniques to avoid this occurrence.

Can you sequence full length IgG?

We do not guarantee full length sequence but more often than not we are able to obtain it. If this is the case we will provide it at no extra cost. In any case we believe that sequencing of the VH and VL domains is sufficient to safeguard an antibody, patent its sequence, and prepare for any future antibody engineering projects.