Scientific Support
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Am I able to obtain the immunogen sequence for this antibody?
Novus selects immunogen sequences in order to deliver the most effective/specific antigenic responses. While the specific immunogen sequences for some products are available on their datasheets, many Novus antibodies are directed against proprietary immunogen sequences. Please contact our Technical Support Team for details.Are there any published references citing the use of this antibody?
Novus strives to keep our product datasheets updated with newly released data and citations. As soon as we receive notification of a newly published reference, our datasheets are promptly updated. The published literature is located at the bottom of each data sheet under the "References" section. Occasionally, authors will cite a Novus antibody without providing a specific catalog number. We will do our best to determine which product was used, however this may require contacting the author for additional clarification.Can I receive a free sample of a product?
Novus does not typically offer free trial sized samples for testing purposes. While some products are offered in sample sizes at reduced prices, not all products may be available in this way. Sample sizes are identifiable by a "SS" at the end of the catalog number. Novus makes every effort to provide our customers with updated data and references in support of our reagents, and we continue to back all of our products with a 100% quality guarantee for the applications and species listed on the datasheet. This policy will provide you with the antibody that you need with no financial risk. In the extremely rare case that the antibody does not work, our technical support team will provide prompt assistance and you will receive your choice of a full refund, credit, or replacement. If the antibody is being used in an untested species or application, then we cannot support it with our guarantee. However, we do encourage that you report your results using our product review survey, available online or by request.Can I use a product in an untested application or species?
Yes! Our Innovator's Reward™ program is designed to support your innovative research with minimal financial risk to you. Should you decide to use one of our products in an untested application or species, Novus will provide you a 50% refund on the purchased product as well as a 50% discount on a future product of equal or lesser value; any product with any result. Simply email innovators@novusbio.com to apply for your award. We will send you a form for you to submit a summary of your research and report your experimental results, after which your award will be given. Your innovation can help us to accelerate bioscience research. After all, we’re making your success our goal.
Can I use this antibody for the detection of an untested species?
Information regarding the known cross-reactivity is usually found under the 'Summary' and 'Species' sections of the product datasheet. If however, you would like to use an antibody in an untested species, then we recommend checking the sequence alignment between the immunogen and the protein of interest. Sequence alignments may be performed using online databases such as Swiss-Prot or ClustalW. An alignment score >85% is a good indication that an antibody may cross-react with the species of interest. However, please note that although an alignment score may be high, Novus is only able to guarantee the antibody’s performance in species stated on the datasheet.Do you have additional information on LIGHTNING LINK CONJUGATION KITS?
Lightning-Link Methodology
* Is the hands-on time really only 30 seconds?
Yes. You will need to familiarize yourself with the instructions before using Lightning-Link(TM) products for the first time; but after that, your hands-on time should be no more than 30 seconds.
Yes. You will need to familiarize yourself with the instructions before using Lightning-Link(TM) products for the first time; but after that, your hands-on time should be no more than 30 seconds.
* How long does the entire conjugation reaction take?
The reaction will be complete after a 3 hour incubation period at room temperature (20 - 25 degrees Celsius), but a longer incubation time will not be detrimental. It is often extremely convenient to set up reactions overnight at room temperature and use the conjugate first thing the next morning.
* What is the ideal starting concentration of the antibody?
Antibody concentration is critical for the Lightning-Link(TM) conjugation reaction, and has been optimized for each kit. Therefore we suggest you consult the relevant instruction manual.
* Does the antibody have to be purified?
Yes. The conjugation chemistry involves free amine groups, therefore any protein present in the mixture will become labeled during the Lightning-Link(TM) reaction. Antibodies which are present in ascites fluid, serum or hybridoma culture media should be avoided. However, purified antibodies are perfect for Lightning-Link(TM) and do not pose any problems.
* What recovery can I expect?
The entire conjugation reaction is contained within one tube. Thus the common losses, such as those on columns during dilutions and upon concentration, simply don't occur. Antibody recovery is nearly 100%.
* How do I separate out free label?
This is not necessary. Lightning-Link(TM) conjugations are highly efficient and the conjugation kits are designed to yield a low level of free label at the end of the reaction; no separation steps are required.
* Do I need to desalt the final conjugate?
Lightning-Link(TM) chemicals are designed to deactivate and are completely benign. In Western Blots, ELISA, IHC, etc., you can use the conjugate right away.
* How scalable is the technology?
Scalability is one of the major advantages of Lightning-Link(TM). Rather than risk valuable antibody you can label a small quantity and be confident that a bulk quantity made later will have essentially identical properties. By avoiding desalting/dialysis steps, the major cause of losses and batch-to-batch variations in conjugation experiments is eliminated.
* Are other quantities available for scale up?
The standard packs address the need for a technology that allows conjugations to be performed on a microgram to milligram scale, but lyophilized material can be provided in any quantity with fast turnaround.
Lightning-Link Antibody Buffers
* What buffers can be used?
Lightning Link(TM) works with phosphate, Hepes, MES and MOPS and other amine-free buffers. Conjugates can also be prepared in the presence of up to 20mM Tris buffer with almost no reduction in coupling efficiency. Once the reaction is complete, the conjugate can be diluted in any buffer that is compatible with both the label and the antibody.
* What buffer additives can be used and what should be avoided?
Additives such as salts (e.g. NaCl), sugars (e.g. sucrose), and chelators (e.g. EDTA) have no effect on the conjugation reaction. The main additives to avoid are nucleophiles; these may deactivate the conjugation chemical which labels the protein of interest. Common nucleophiles used in laboratories include amino acids (e.g. glycine), blockers (e.g. ethanolamine) and thiols (DTT, mercaptoethanol).
* The antibody storage buffer contains common additives such as Bovine Serum Albumin (BSA) or gelatin. Will this effect conjugation chemistry?
Concentrations of up to 0.5% BSA and 0.1% Gelatin have little effect on the conjugation chemistry.
* Does sodium azide cause interference?
A slight reduction in efficiency has been observed in head-to-head trials for samples with and without azide, but for all practical purposes this is of little significance.
* How do I remove additives from the antibody storage buffer, which are at a higher concentration than that recommended for the Lightning-Link reaction?
The AbSelect(TM) antibody purification kit enables you to remove such additives with ease. Most importantly, the components of the AbSelect(TM) purification kit are fully compatible with the Lightning-Link(TM) conjugation system. This means that the purified antibody can be added directly to any of the Lightning-Link(TM) kits.
* Do I need to add preservatives?
Lightning-Link(TM) conjugates are just like any other conjugates. For long-term storage at 4 degrees Celsius, you may wish to add antimicrobial agents or stabilizers (e.g. azide, BSA, glycerol, etc.)
Lightning-Link Conjugation Chemistry
* What functional groups do I need on my protein to use Lightning-Link?
Lightning-Link(TM) requires amine groups on the molecule to be labeled. Most proteins or peptides have lysine and/or alpha-amino groups. All antibodies have multiple amine functions.
* Can proteins other than antibodies be labeled?
Yes. While labeling of primary antibodies is a major application, the only requirement is that the protein or molecule to be labeled has amine functionality.
* Is the label attached to my protein by a covalent bond?
Yes. Conjugates formed with Lightning-Link(TM) are perfectly stable.
* Will my antibody couple to itself or form high molecular weight aggregates?
No. Although Lightning-Link(TM) is a one-step conjugation method it has no similarity to other simple methods (e.g. glutaraldehyde) which tend to give ill-defined aggregates.
* Is my protein exposed to high pH?
No. Unlike conventional conjugation methods, Lightning-Link(TM) does not expose molecules to high pH. The conjugations are carried out at physiological pH.
Do you have info about using HIF-1 alpha in Western Blot?
- The HIF proteins are among the most rapidly degrading proteins ever studied. Upon cellular re-oxygenation it can be completely degraded in less than 1 minute. Therefore, it is critical to prep only a few plates/dishes/flasks of cells at a time and to immediately place the cells into ice cold buffers and perform the whole protein prep on ice.
- HIF-1 is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5% or with treatment using certain agents (CoCl2, DFO, etc.) so proper sample preparation is critical.
- Upon stabilization HIF-1 translocates to the nucleus. The best western blots (cleanest) are always done using nuclear extracts. It is possible to detect HIF-1 in whole cell extracts, but they tend to be much dirtier and the staining is much weaker.
- Finally, we recommend that a positive/negative control always be run side by side so that it is possible to discern which band is upregulated in the hypoxic sample. Unprocessed HIF1 is ~95 kDa while the fully post-translationally modified form is ~116 kDa, or larger. Additionally, HIF-1 alpha may form a heterodimer with HIF-1 beta (Duan, et al. Circulation. 2005;111:2227-2232.). Depending on the sample, treatment, etc. you may see either a band or a doublet.
Does Novus Biologicals make Custom Antibodies?
No, but our partner SDIX does! SDIX offers a full suite of custom services to successfully create an antibody for your research needs. Their team of experts will work with you closely to cater to your needs. Within SDIX's Custom Antibody Services, you will find:
- Technical expertise and strategy development
- Custom antigen solutions, including peptide, recombinant proteins, and new technologies such as the SDIX proprietary Genomic Antibody Technology (see below)
- Custom immunization protocols
- Custom screening strategies
- Various production scales
Genomic Antibody Technology, or GAT, is a complete process that takes you from protein sequence to the delivery of a purified antibody ready for use. To learn more about this specific immunization process, click HERE.
Find more details about SDIX's Custom Antibody Services by clicking HERE or by emailing antibodies@sdix.com.
Has product X been tested in species Y or application Z that is not listed on the data sheet?
If you do not see that a specific species or a specific application is listed on our data, please contact our Technical Support Team and they will be happy to check their reference database to see if any other researchers or publications have cited the use of this antibody in the species or application of interest.How do I select an isotype control for my experiments?
When purchasing an 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, 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 mouse. Where applicable, the isotype control should also be conjugated to an identical fluorochrome as the primary antibody being tested. Recommended isotype controls can usually be found in the products drop-down menu on the Novus Biologicals website.How should I aliquot my antibody?
In order to avoid repeat freeze thaw cycles, Novus recommends aliquotting the antibody prior to storage below freezing. Aliquots should be no smaller than 20 ul as the antibody can absorb into the sides of the vials. Alternatively, you may dilute the contents with a solution containing 1:2 – 1:20 BSA. The additional proteins act to stabilize the antibody solution. Before diluting the antibody for storage, however, the diluent should be sterile filtered to retard microbial growth.How should I choose a positive control?
Novus strongly recommends testing antibodies in the presence of suitable controls. Our product datasheets will often list the suggested positive control tissues, cell lines, or extracts. However, for products where this information is not listed, we recommend searching for known positive controls in many popular online protein databases (i.e. Swiss-Prot, NCBI, GeneCards, ATCC).Additionally, you may always contact our Technical Support Team for details.How should I store my antibody?
Novus always recommends storing the antibody as directed on the datasheet. Improper storage may result in loss of antibody activity, so Novus is unable to guarantee an antibody’s performance if not stored as recommended. Antibodies are adversely affected by freeze/thaw cycles. Therefore, it is typically recommended to store antibodies for a few days at 4° C rather than expose them to multiple freeze/thaw cycles. Do NOT store antibodies in frost-free freezers.How should I choose a suitable secondary antibody?
Secondary antibodies should be raised against the host species of the primary antibody that you are using. For example, if your primary is a chicken polyclonal IgG, then an anti-chicken secondary should be used. We recommend that you check the datasheet of the secondary antibody to ensure it has been validated for the application you will be using. For multiplex assays using primary antibodies from different species, be sure the secondaries have been pre-absorbed for cross-reactivity. Novus provides a wide range of secondary antibodies in various conjugation formats, and you may always contact our Technical Support Team for assistance in choosing the best option.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.What is SDIX Genomic Antibody Technology™?
Novus Biologicals is the exclusive worldwide distributor of SDIX's premade Genomic Antibody Technology™ (GAT) polyclonal antibodies. What exactly is SDIX Genomic Antibody Technology™? Read on to learn more or download the SDIX GAT™ White Paper here.
SDIX GAT™ uses sophisticated bioinformatics and immunization strategies to produce high-value antibody reagents and biomolecules. SDIX’s application of powerful proprietary algorithms enables SDIX GAT to focus antibody creation on a precise gene or protein sequence that directs the resultant antibody to specifically bind to that region of the protein in its naturally folded form. The ability of any antibody to recognize a protein’s naturally folded state has the potential to expand an antibody’s utility to high-value applications like sandwich immunoassay, ChIP, and flow cytometry. Another advantage of this technology is the ability to produce reagents against traditionally difficult cellular targets, such as highly conserved and transmembrane proteins.
Please contact the Novus Technical Support team at technical@novsubio.com if you have questions regarding SDIX GAT™ and the SDIX GAT™ polyclonal antibodies.
Why does NB100-105 detect a target at 120KDa when according to Swiss Prot the two isoforms run at approximately 80-90 KD?
Hif-1 alpha's predicted molecular weight is approximately 97kDa. However, the protein is undergoes significant amounts of post-translational modification which results in a protein which migrates at 116 kDa as is reported throughout the primary literature. Depending on the treatment used to upregulate HIF-1 (i.e true hypoxia vs. hypoxic mimics like CoCl2 or DFO) the relative amounts of the different species of the protein can vary significantly. For instance treatment with CoCl2 will often result in large amounts of the 97 kDa species and little of the 116kDa species, while true hypoxia often results in the opposite. This is why it is of critical importance to always run treated and untreated samples in adjacent lanes so as to be able to compare the induction of HIF-1 in the treated sample. Note also that the Isoform 2 listed in Swiss-Prot has only been inferred from sequence and has not been demonstrated experimentally
Why has this antibody been discontinued and is there a way to obtain the discontinued stock?
Occasionally, it becomes necessary to discontinue a product from our catalog. Some reasons include a lack of commercial interest, a disruption in supply from an outside collaborator, or unforeseen production difficulties. Rarely, a product will be discontinued for quality issues. We understand that this can be an inconvenience to our customers, and we will do our best to recommend an appropriate alternative, when available. Please contact our technical support team for help in finding any potential replacement products. Once we list an antibody as unavailable on our website, the remaining stock has been exhausted and no additional vials remain.Why is the actual Western blot band size different from the predicted?
In general, as proteins migrate through a PAGE gel matrix, they are separated according to their size (MW). The smaller the protein, the faster it migrates through the gel. However, migration may also be affected by other factors. Therefore, the actual band size observed may differ from the predicted size. Some of these factors include post-translational modification, post-translational cleavage, splice variation, relative charge variation, and the forming of protein multimers.Why is the antibody concentration unavailable for some ascites (monoclonal) or antisera (polyclonal) products?
It is not possible to determine the concentration of these antibodies, as unpurified ascites and antisera contain serum proteins/immunoglobulins other than the antibody of interest. Therefore, an exact antibody concentration is usually only relevant for purified antibodies.