Technical Support: FAQs

1. Storage and Shipping

2. Concentrations and Purification

3. Applications

4. Conjugates

5. LYNX Rapid Conjugation Kits®

6. Secondary antibodies and Isotype controls

7. Antibody Specificity

8. Custom Services

9. Technical Services

What is the concentration of my AbD Serotec antibody?

The antibody concentration usually appears on the product datasheet. If this information is absent then please consider the following.

We do not measure the exact amount of specific antibody present in certain antibody preparations including tissue culture supernatants, serums and ascites. The table below gives an approximation of the amount of specific antibody that can be expected in such preparations.

Format Approximate specific antibody concentration
Tissue culture supernatants 10 – 50 ug/ml specific antibody
Serum 0.5 -1 mg/ml specific antibody
Ascites 1-5 mg/ml specific antibody

If the antibody is supplied in a ready-to-use format then it is exactly that. The concentration of each batch will be optimized for the application indicated on the datasheet (generally immunohistochemistry) and may vary slightly between batches. An approximate concentration of 1-5 µg/ml can be assumed.

Similarly, if the antibody is supplied as a number of tests then the concentration of each batch will be optimized for the application indicated on the datasheet (generally flow cytometry). If you wish to incorporate an isotype control into your experiment, and therefore require further advice on the concentration of your test antibody, then please contact Technical Support for guidance.

What concentration of antibody do I need to use for my experiment?

As stated on our product datasheets, supported applications may be derived from testing within our laboratories, peer-reviewed publications or via personal communication with the antibody originator(s). Suggested dilutions may not be available for all applications. Under such circumstances please refer to the general recommendations given in the table below.

Application Approximate working concentration* Additional Notes
Flow cytometry 10 ug/ml Use 10 ul of antibody at 10 ug/ml to label 100 ul of whole blood or 10(6)cells.
Immunohistochemistry 10 ug/ml Use approximately 100 ul/slide ensuring tissue sections are completely covered.
Immunoprecipitation 10-50 ug/ml  
ELISA Coating: 1-10 ug/ml
Detection: 1-5 ug/ml 		For a 96 well plate, 100 ul is optimal.
Western Blotting 1 – 10 ug/ml <1 ug/ml may be necessary when using a sensitive detection system.
Blocking experiments 1-10 ug/ml  

*Concentrations are given as a guide only. It is always recommended that the user titrates the antibody for use in their own system using the appropriate negative/positive controls.

The approximate working concentrations outlined in the table above can also be used as a starting point where the suitability of an antibody for a given application has not been determined. Please note, AbD Serotec is not able to guarantee the suitability of antibodies for applications which are listed on datasheets as “Not determined”.

Will my AbD Serotec antibody work in an application of interest which is listed in the applications table as “Not Determined”?

The antibody may work but, unfortunately, we cannot offer any guarantees because (i) to our knowledge, it has not been tested or (ii) we do not have strong enough evidence of a successful result. You may like to contact Technical Support to inquire whether or not we have additional information on file e.g. a customer report. Alternatively, if the product of interest is a monoclonal, then a search of the literature/internet using the clone name may throw up additional information which has escaped our attention. The clone name can be found on the product datasheet.

Will my AbD Serotec antibody work in an application of interest which is listed in the applications table as “No”?

We do not list a product as being unsuitable for a particular application unless we have evidence to indicate that it has been tested without success. Of course it is always possible that in another researcher’s hands, under slightly different working conditions, different results might be obtained.

How should I store my AbD Serotec antibody?

Recommended storage conditions can be found on all product datasheets. Always refer to the datasheet for the most up-to-date information. Generally, if the product does not contain any kind of preservative e.g. sodium azide or thimerosal then we will recommend storage at -20ºC.

If preservatives are present then datasheets usually recommend storage at +4ºC or -20ºC if preferred. Lyophilized products, which do not contain preservative can usually be stored at +4ºC until reconstitution but should be stored at -20ºC thereafter. RPE conjugates must NOT be frozen.

My AbD Serotec antibody was shipped without ice but the recommended storage conditions are listed as +4ºC or -20ºC, will the activity have been affected?

No. AbD Serotec ship thousands of antibodies every week, to customers all over the world, at ambient temperature. Loss of activity has never been a problem. However, on receipt, antibodies should immediately be stored in accordance with the datasheet recommendations.

My AbD Serotec product arrived today but the dry ice had evaporated, will the activity have been affected.

Antibodies are very robust. It is very unlikely that the activity will have been affected during the time period concerned. However, in the unlikely event that the antibody fails to perform as expected, please do not hesitate to contact Technical Support. If your product is not an antibody please contact us immediately for further advice.

What is the expiry date of my AbD Serotec antibody?

Information relating to the shelf life of any given product can be found on the product datasheet. For most products shelf life is given as 18 months from the date of despatch or (for lyophilized products) 12 months from the date of reconstitution. Always refer to the product datasheet for the most accurate information. We are able to provide a shelf life in this format as we carefully control storage conditions of all products before shipping from our facilities.

What is the amino acid sequence of the epitope recognized by my AbD Serotec monoclonal antibody?

AbD Serotec do not carry out epitope mapping of our antibodies and for the vast majority of products this information will not be available. This is particularly true for antibodies raised against a cell type rather than a known peptide sequence. Any information that we have is likely to be based on the literature and will appear on the product datasheet.

Can I use my AbD Serotec antibody for in vivo experiments?

AbD Serotec have not tested and therefore are not in a position to recommend any of our products for in vivo animal experiments. In the UK, such studies are heavily regulated by The Home Office.

Can I use my AbD Serotec antibody for in vitro experiments?

Where identified, information relating to functional activity will be included on the product datasheet. AbD Serotec recommend the use of preservative free, Low Endotoxin formats for in vitro experiments. If such a format is unavailable then azide can be removed using a dialysis cassette (EQU003).

Can I use my AbD Serotec antibody for diagnostic purposes?

The vast majority of AbD Serotec products are for research purposes only although we do offer the serotec FL™ range, a group of CE marked antibodies specifically for diagnostic use.

We do not currently offer any products certified by the FDA as IVD (or ASR), therefore all of our products sold in North America are supplied as Research Use Only.

Can AbD Serotec make me an antibody against my target of interest?

Yes. AbD Serotec uses proven HuCAL PLATINUM recombinant antibody technology to custom develop highly-specific monoclonal antibodies in just 8 weeks. Please refer to our website for further information.

How was my AbD Serotec monoclonal antibody purified?

Most AbD Serotec monoclonal antibodies of IgG class are purified using Protein G or Protein A affinity chromatography. This results in antibody preparations of >95% purity.

Monoclonal antibodies of the IgM class are generally purified by a combination of ammonium sulfate precipitation and/or size-exclusion chromatography.

How was my AbD Serotec polyclonal antibody purified?

Many AbD Serotec polyclonal antibodies are purified using antigen-affinity chromatography. This results in an antibody preparation of at least 90% antigen specific antibody, providing high titers and minimal cross-reactivity with other proteins.

Some polyclonal antibodies are purified using Protein A or Protein G chromatography, ion exchange chromatography, or by ammonium sulfate precipitation. In these cases the total IgG content is usually >90%, however due to the nature of polyclonal antibodies, it is likely that the specific antibody content will be approximately 10%.

How does AbD Serotec measure the immunoglobulin concentration of its purified antibodies?

Immunoglobulin concentration is measured by absorbance at OD280nm. An extinction co-efficient of 1.43 is used i.e. a 1 mg/ml solution will give an OD reading of 1.43.

How do I select a secondary antibody for my experiments?

There are many things to consider when purchasing a secondary antibody including the host in which your primary antibody was raised, the species in which you are working, the application in question, and the preferred detection system. Recommended secondary antibodies can usually be found at the bottom of AbD Serotec product datasheets. If this information is absent, or your primary antibody was not purchased from AbD Serotec, then please see our online guide or contact Technical Support for advice.

What is an isotype control?

All immunoglobulins will bind non-specifically to cells expressing Fc receptors on their surface. Antibodies raised in mice, particularly of the IgG2a isotype, bind strongly to some human leukocytes. Isotype or negative controls are incorporated into flow cytometry (or occasionally IHC) protocols to assess the level of non-specific binding to Fc receptors on the target cell.

How do I select an isotype control for my experiments?

When purchasing a isotype or negative control for your experiment, in addition to the host species and isotype of your test antibody, you will also need to consider the species in which you are working. An isotype control may be raised against an antigen that is not naturally occurring e.g. DNP but this is not always the case. Please be aware that an isotype control advertised as being suitable for use on human cells may not be suitable for an experiment where the target cells are rat.

Where applicable, the isotype control should also be conjugated to an identical fluorochrome as your test antibody e.g. whilst spectrally similar, an APC-conjugated test antibody should not be paired with an Alexa Fluor 647®-conjugated isotype control.

Recommended isotype controls can usually be found at the bottom of AbD Serotec product datasheets. If this information is absent, or if your test antibody was not purchased from AbD Serotec, then please contact Technical Support for further advice.

What if an AbD Serotec reagent does not work as expected?

At AbD Serotec we work hard to ensure that all our antibodies perform well and consistently, and go to great lengths to make sure our datasheets are easy to read, clear and unambiguous. However, we recognize that there is always scope for improvement. In the unlikely event that you find an AbD Serotec reagent not working as described on our datasheet, then please contact Technical Support.

We have a fair and objective policy for troubleshooting and our procedure is designed to provide you with the quickest possible resolution. If any AbD Serotec product is found to be faulty at any time during its guarantee period, we will offer you the choice of an immediate replacement or a credit note.

If you do encounter a problem with any AbD Serotec product please notify your local AbD Serotec office or distributor. If immediate troubleshooting is unsuccessful you will receive a detailed troubleshooting form for completion. This form allows us to collect all the information that is needed to make an accurate assessment of the problem.

Please complete the troubleshooting form as fully and as quickly as possible, and return it to your AbD Serotec contact. On some occasions it may be necessary for you to attach further details or example results on additional sheets. We are always happy to help you with any questions you may have concerning the completion of this form. The more information you can provide at the start of this process the better, as this allows us to investigate as efficiently as possible.

I can’t find the antibody that I’m looking for? Is it available from AbD Serotec?

If you are struggling to find what you are looking for, our Antibody Detectives are always happy to help. We’ll assign you a personal Detective to track down your antibody for free – even if it’s from an alternative commercial source.

I can’t find the product that I’m looking for? Has it been deleted?

It may have been deleted but alternatively it may have undergone a format change. For example, a tissue culture supernatant format may have been upgraded to a purified format. In this situation the product will have been reintroduced under a new catalog code. Our website should re-direct you automatically, but if not please contact Technical Support for further assistance. If the product you require is no longer available then we will try and find you an alternative AbD Serotec product, or suggest an alternative commercial source.

You have the antibody specificity that I want but it’s not available conjugated to the fluorochrome of interest. Where do I go from here?

We may be able to meet your needs via our Custom Conjugation Service. Please contact Technical Support or visit our website for further information.

Please note that certain fluorochromes we conjugate directly to our primary antibodies are under patent and, as such, cannot be offered for custom conjugation. However, there may be spectrally similar dyes within the AbD Serotec range that will meet your requirements.

What is the difference between a Western blot performed under reducing versus non-reducing conditions?

To perform a Western blot under reducing conditions simply include a reducing agent such as dithiothreitol (DTT) or ß-mercaptoethanol in the SDS sample buffer. These reducing agents act to disrupt any disulfide bonds. The 150 kDa IgG molecule, for example, will be separated into 2x heavy (~50 kDa) and 2x light chains (~25 kDa).

Should I use reducing or non-reducing conditions for my Western blotting?

Western blotting is most commonly run under reducing conditions. If this information is not listed on the product datasheet then it may not be available to us. Please feel free to contact Technical Support as we may have additional information on file. Alternatively, if the product of interest is a monoclonal, then a search of the literature/internet using the clone name in question may throw up helpful references which have escaped our attention. The clone name can be found on the product datasheet.

What is the FITC:protein ratio of my AbD Serotec antibody?

This information is batch-specific and not listed on our product datasheets. It is usually between 4:1 and 7:1. If you require a more accurate figure then please contact Technical Support with the relevant batch details.

What is the RPE:protein ratio of my AbD Serotec antibody?

The RPE:protein concentration is usually 1:1. Antibodies for conjugation to RPE are affinity purified prior to conjugation. They are subsequently purified by size exclusion chromatography. Those with a RPE:protein ratio greater than 1:1, unconjugated antibody and unbound RPE molecules are discarded during this process.

What is the biotin:protein ratio of my AbD Serotec antibody?

We do not measure the biotin:protein ratio of our antibodies. Biotin is conjugated via lysine groups and the product is extensively dialyzed. It can therefore be assumed that there is no free biotin present.

What is the APC:protein ratio of my AbD Serotec antibody?

The APC:protein concentration is usually 1:1.

What is the RPE-Cy5:protein ratio of my AbD Serotec antibody?

The RPE-Cy5:protein concentration is usually 1:1.

What is the Alexa Fluor® dye:protein ratio of my AbD Serotec antibody?

This information is batch-specific and will not appear on the product datasheet. If you require information relating to the degree of labeling then please contact Technical Support with the relevant batch details.

What is the Pacific Blue™:protein ratio of my AbD Serotec antibody?

This information is batch-specific and will not appear on the product datasheet. If you require information relating to the degree of labeling then please contact Technical Support with the relevant batch details.

What is the Dylight™ dye:protein ratio of my AbD Serotec antibody?

This information is batch-specific and will not appear on the product datasheet. If you require information relating to the degree of labeling then please contact Technical Support with the relevant batch details.

What is the HRP:protein ratio of my AbD Serotec Antibody?

This information is batch-specific and will not appear on the product datasheet. A ratio of 1:0:1.2 (+0.6) is considered acceptable. If you require a more accurate figure then please contact Technical Support with the relevant batch details.

Can AbD Serotec recommend an antibody specific for the cell type that I am studying?

This is not a straightforward request. We may be able to make suggestions based on published literature but very few markers are entirely specific for one cell type. As a researcher in the field, you probably know far more about the specific cell type that you are studying than we do.

Can Leucoperm™ (BUF09) be used for staining intracellular markers alongside cell surface staining?

Yes, but staining of the cell surface antigen (preferably with a directly-conjugated antibody) must be performed prior to fixation/permeabilization with Leucoperm™ and the subsequent intracellular staining.

Can Leucoperm™ (BUF09) be used for staining intracellular markers by indirect flow cytometry?

Yes, but AbD Serotec always advises the use of a direct conjugate where available. The use of a secondary antibody may result in a higher level of background staining. If intracellular staining is preceded by the staining of a cell surface antigen then particular care must be taken when selecting a secondary antibody.

Are RPE-conjugated antibodies too big for intracellular staining protocols?

No, RPE conjugated antibodies can be used to label intracellular antigens in conjunction with Leucoperm™ (BUF09). An example histogram illustrating successful intracellular staining with an RPE-conjugated monoclonal antibody together with Leucoperm™ can be seen on the product datasheet for MCA341PE.

Alexa Fluor® is a registered trademark and Pacific Blue™ is a trademark of Molecular Probes Inc, OR, USA.

DyLight™ is a trademark of Thermo Fisher Scientific and its subsidiaries.

Leucoperm™ is made for AbD Serotec by AN DER GRUB Bio Research GmbH

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