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 this antibody be used for in vivo experiments?
Novus Biologicals antibodies are intended for use in vitro experiments only. Our antibodies have not been tested nor are recommended for use in vivo.
Can I use this antibody 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 reward you a discount voucher for 100% of the purchase price of the reviewed product. Simply email email@example.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. You can also submit a review detailing your positive/negative results for the untested species/application. Novus will verify the image data and process the review. We will email you a voucher for 100% of the purchase price of the reviewed product to use for a future purchase and you will receive rewards. Your innovation can help us to accelerate bioscience research. After all, we’re making your success our goal.
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.
- 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.
- Novus offers hypoxic and nomoxic cell lysates for positive controls in your western blot experiments. Both CoCl2 induced and true hypoxia induced cell lysates are available.
“EPO transcription can be activated by exposure of Hep3B cells to either hypoxia or cobalt chloride (7). HIF-1 binding activity was induced after 1 h and was maximal after 4-h treatment of Hep3B cells with 75 uM cobalt chloride (Fig. 2A), which is similar to the kinetics of HIF-1 induction by hypoxia (data not shown). Exposure of HeLa cells to cobalt chloride for 4 h also induced HIF-1 activity. In contrast to hypoxia, which induced a doublet band corresponding to HIF-1 in EMSAs, cobalt chloride induced a single band of HIF-1 activity in both Hep3B and HeLa cells (compare Figs. 1A and 2A). We have not determined the basis for this reproducible difference in response to stimulation by hypoxia as compared to cobalt chloride” (Wang G, et al. (1993) PNAS 90, 4304-4308.). Thus, it is critical to be able to look at upregulation compared to the control.
Does Novus Biologicals make Custom Antibodies?
No, but Bio-Techne does! Since July of 2014, Novus Biologicals becomes a part of the Bio-Techne team to broaden our portfolio of products, and therefore supports custom services on producing specialized antibodies. Bio-Techne Custom Service provide the following technical areas:
- Antibody of interests
- Custom recombinant proteins
- Specialized ELISA platforms
- Highly specific Luminex assays
- Biomarker testings
Bio-Techne is a leading developer and manufacturer of purified proteins, which is the new trade name for Techne Corporation, the parent company of life science and diagnostic brands R&D Systems, Tocris, Novus Biologicals, ProteinSimple, and BiosPacific.
Find more details about Bio-Techne Custom Services for the variety of biological products including antibodies, please contact Kathryn Fellis, Technical Sales Consultant at 415.418.8283 or by emailing firstname.lastname@example.org.
Is the GST tag at the n- or c-terminus on Abnova proteins?
GST tag is at the N-terminal and it could be cleaved from the fusion protein by PreScission Protease (Amersham Biosciences).
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.
What antigen retrieval method should I use?
Antigen retrieval information is not always available for all immunohistochemistry antibodies, so some optimization by the end user is often required. Please refer to the Antibody Guide for more information on antigen unmasking procedures.
What is the concentration?
The concentration for our products is listed on the online datasheet. Simply search for the catalog number in which you are interested, and view on the “Datasheet” tab, under the "Applications" section. The concentration is also listed on the printed datasheet you received with your product. You may also contact our Technical Support Team at email@example.com or by calling 1-888-506-6887.
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 firstname.lastname@example.org if you have questions regarding SDIX GAT™ and the SDIX GAT™ polyclonal antibodies.
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 the molecular weight of IgG?
An IgG protein is comprised of two heavy chains and two light chains. The heavy chains are approximately 55 kDa each and the light chains that are approximately 25 kDa each. Combined, this equals a total molecular weight of approximately 160 kDa.
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.
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.
Your biomolecule should be in a sitable, 10-50mM amine free buffer (e.g. MES, MOPS, HEPES, PBS), pH range 6.5-8.5 and not in ascites fluid, serum or tissue culture supernatant. It should not contain any additives such as Azide, BSA, Tris or Glycine at a concentration of >0.1%.
The amount of antibody (IgG) you should add to the Lightning-Link® vial usually corresponds to the kit size you purchased (for example: a 3x100µg kit enable you to label 3 lots of 100µg antibody) and the volume added should also match (eg: 100µl), meaning the ideal concentration is 1mg/ml.
NOTE: The amount and volume of antibody recommended above are for all Lightning-Link® kits, with the exception of RPE and PE tandem dyes. For more details on the recommendations specific to these dyes, please consult the protocols available on each product page.
- How long does the entire conjugation reaction take?
- When using standard Lightning-Link® kits the reaction will be complete after a 3 hour incubation period at room temperature (20-25oC), although a longer incubation time will not be detrimental. Lightning-Link® Rapid kits have an incubation time of just 15 minutes. DyLight dyes are only available in the Rapid format.
- Can I label a peptide / protein other than IgG?
- Yes. The Lightning-Link® technology works by targeting free amine groups on your target, meaning it can be used to label most biomolecules.
- Do my antibody and buffer meet the requirements for labelling?
- Your antibody should be purified, as other molecules with free amine groups will also become labelled, resulting in a poor quality conjugate. A suitable method of purification should be used (such as affinity or Protein A/G). Please note that 0.2/0.22 µm filtration is a method of sterilisation, not purification
- What can I do if my antibody formulation does not meet the requirements?
- If your antibody buffer is not compatible with the Lightning-Link® kits, you may wish to use an AbSelect™ purification kit. These kits allow you to quickly and simply purify your antibody and they are fully compatible with the Lightning-Link® kits. The choice of AbSelect™ kit depends on your particular sample (species, buffer, contaminants, volume). Please feel free to contact us if you require further guidance.
If your antibody is already purified but its concentration is too low, you can concentrate it by using an Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, Tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link®.
If your antibody contains BSA, you can use a BSA removal kit to purify your antibody in a few simple steps. Please note that this kit will also enable you to concentrate your antibody.
NB: The AbSelect™ kits will only work with antibodies, and they will not purify other molecules. The Antibody Concentration and Clean Up Kit will work with other molecules greater than 10kD.
- What recovery can I expect?
- The entire conjugation reaction is contained within one tube. Thus common losses, such as those associated with the use of columns, simply don't occur. Antibody recovery is nearly 100%.
- How do I separate out free label?
- This is not necessary. Lightning-Link 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. Any remaining free label would have its reactive 'Lightning-Link®' groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
- How many labels will bind to my biomolecule?
- It is difficult to give a precise answer to this as the result will vary from antibody to antibody (or other protein).
- How stable will my new conjugate be?
- The Lightning-Link® chemistry joins the label to the antibody via a stable, covalent bond. The stability of your conjugate will therefore be dependent on the stability of your antibody, and on your label of choice.
- How should I store my new conjugate?
- The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. A new conjugate can be stored at 4°C as long as the antibody will tolerate storage at 4°C. As the bond between the antibody and dye is covalent and very stable, it should not degrade; therefore at 4°C no additional preservatives are needed. Storing the conjugate undiluted is recommended. We suggest that the optimal storage conditions for any conjugate are determined experimentally, using small aliquots of the conjugate. Note that the preservative sodium azide will inhibit HRP.