Antibody and Western blot antigen standard FAQs
What makes OriGene the optimal source for antibodies?
Answer: All of OriGene’s antibodies have been validated by Western blot analysis and are guaranteed to detect the protein of the predicted molecular weight in the antigen standard provided. In addition, a positive control (an aliquot of a cell lysate with the corresponding antigen overexpressed) is included with every antibody order. The customers have the money back guarantee for antibody performance.
What are the terms of OriGene’s antibody guarantee?
Answer: We guarantee that our validated antibodies will detect the corresponding protein in the gene specific Western blot antigen standard provided with each antibody. If you encounter any difficulty detecting the antigen in OriGene’s VERIFY overexpression lysate, our Technical Services professionals will help troubleshoot and optimize your experimental conditions.
How can OriGene offer this quality guarantee?
Answer: OriGene has the world’s largest collection of full-length, expression-ready cDNA clones. We have used this vast collection to generate overexpression cell lysates by transiently transfecting HEK293T cells with the TrueORF cDNAs. This creates gene-specific myc and DDK tagged antigen standard that serve as high-quality validation tools for all OriGene antibodies by Western blot analysis. Only antibodies that pass our stringent quality control experiments and meet our high standards for specificity and sensitivity are provided.
How can OriGene ensure that the antibody I’m interested in detects the endogenous, overexpressed or down-regulated protein?
Answer: OriGene only sells antibodies that detect the protein at the predicted molecular weight from a gene-specific antigen standard
Does OriGene guarantee that the antibody will detect the endogenous or overexpressed protein prepared by the customer?
Answer: No. Since OriGene cannot confirm that your cell line, tissue or overexpression construct produces the specific protein at levels detectable by Western blotting, we only guarantee positive detection of the included antigen standard using our validated antibody.
How do OriGene’s antibody prices compare to other companies’ prices?
Answer: On average, OriGene’s antibodies and our Western blot reagent cost less than our competitors’ products but guarantee the highest quality. At the same time, we also offer a free vial of VERIFY tagged overexpression lysate for antibody validation.
Does OriGene sell validated overexpression Lysates separately?
Answer: Yes. We sell bigger package of VERIFY tagged overexpression lysate separately. Please see our website for a list of available lysates that can be purchased separately.
Are the antibodies monoclonal or polyclonal?
Answer: For the gene-specific antibodies, we offer both monoclonal and polyclonal antibodies. The detailed information can be obtained from Origene’s antibody webpage. All our anti-tag antibodies are monoclonal (mouse) and our HRP conjugated secondary antibody is polyclonal anti-mouse antibody that is made from goat (TA100015).
Do your antibodies work in immunohistochemistry or immunofluorescence experiments? Will they work with formalin fixed paraffin embedded (FFPE) samples?
Answer: Each antibody shows different coverage of applications. Customer can find the application for each particular antibody by visiting Origene antibody website.
What source do you use for the predicted molecular weight of the antigen corresponding to each antibody?
Answer: We mainly use the swissprot webpage to help predict the molecular weight of unprocessed protein. Please see the following link: http://www.expasy.org/sprot/
What are the terms of OriGene’s Western detecting reagents guarantee?
Answer: We guarantee that our horseradish peroxidase (HRP) conjugated anti-mouse secondary antibody (TA100015) in conjuction with Western Blotting Luminol Reagent (TA100016) will detect the corresponding monoclonal anti-Tag antibodies used in Western blot analysis. If you encounter any difficulty detecting the antigen in OriGene’s VERIFY overexpression cell lysate, our Technical Services professionals will help troubleshoot and optimize your experimental conditions.
Do you have isotype controls available?
Answer: No. As we offer the validated antibodies only, we do not think it is necessary to include such kind of control in the Western blotting. However, if wanted, you can purchase the control immunoglobulin from other companies.
How should I choose a suitable secondary antibody?
Answer: The selection of secondary antibody is totally dependent on the primary antibody you are using. The rule of thumb is that the secondary antibodies should be raised against the host species as the primary antibody you are using. For instance, if your primary antibody is a mouse monoclonal, you will need an anti-mouse secondary. Origene provides secondary antibodies conjugated to HRP (TA100015) for ECL analysis.
What dilution of primary antibody should I use?
Answer: The datasheet for most of the Origene antibodies should have a dilution suggestion. Please remember that these dilutions and concentration estimates are simply recommended as a starting point, and it may be necessary to adjust the dilution based on the experimental results. In case of no such information provided, the following table can be used as a rough guideline for initial start point.
||Tissue culture supernatant
|WB / dot blot
|IHC / ICC neat
||neat - 1/10
||1/50 - 1/100
|EIA / ELISA
|FACS / Flow Cytometry
||1 - 10 ug/ml
Why is the actual western blot band size different from the theoretical molecular prediction?
Answer: In Western Blotting analysis, the migration of proteins on the gel is based on their molecular weight. However, under certain circumstances, there is a discrepancy between the actual western blot band size and predicted molecular weight. The following factors are the common reasons for this.
- Post-translational modification - e.g. phosphorylation, glycosylation etc, which increases the size of the protein.
- Post-translation cleavage - e.g. many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases
- Splice variants - alternative splicing may create different sized proteins produced from the same gene
- Relative charge - the composition of amino acids (charged vs non-charged)
- Multimers - e.g. dimerization of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands
How should I store my antibody?
Answer: After receiving the antibody, the customer should store the antibody according to the instructions as directed on the datasheet. We are unable to guarantee how the antibody will perform if not stored as stated on the datasheet.
How should I reconstitute the VERIFY tagged antigen standard for antibody validation?
Answer: For Western Blot validation, the overexpression lysate sample can be diluted with 2xSDS sample buffer. After dilution, the lysate sample should be stored at -80℃ for long term storage. Prior to SDS-PAGE fractionation, add reducing agent (100mM DTT or 2.5% ß-ME) freshly and heat the sample at 70℃ for 10 minutes before loading.
How much lysate sample shall I use for each loading?
Answer: Since the VERIFY tagged antigen standard is the gene specific overexpression lysate, the optimal SDS-PAGE loading amount need to be determined by preliminary experimental data from customer. Based on our experience, we suggest customer dilute the lysate with 20ul of 2xSDS loading dye and load around 5ul for one lane.
Can I use the included antigen standard for immunoprecipitation and other experiments?
Answer: The antigen standard is supplied in liquid format in RIPA buffer. One can use our standard for immunoprecipitation, ELISA standard, protein/protein interaction, etc.
Q: Why does my antibody detect multiple bands with your antigen standard?
A: There are several possible explanations for this result and they are not mutually exclusive. First, each lysate is prepared from Hek293T cells and every molecule of the expressed antigen may be subject to various types and degrees of post-translational modifications (e.g. glycosylation, ubiquitinylation, acetylation) thereby increasing the number of bands detectable via W.blotting. In addition, proteolytic degradation can occur despite the presence of protease inhibitors in our extraction buffer. In these cases, our anti-DDK antibody will only show the segment(s) of the protein that retains the DDK tag. A protein-specific antibody might detect other fragments. Also, some membrane proteins tend to form aggregates during SDS-PAGE electrophoresis, or during sample preparation and will run at very high molecular weights. One other possibility is antibody detection of both endogenous protein and myc-DDK tagged over expression protein at slightly different molecular weight
Q: I need to cite your product for a paper I am writing. What language should I use?
A: We recommend that you refer to the product by its specific catalog number and refer to us as OriGene Technologies (Rockville, MD). Furthermore, we'd love to hear from you when your paper is published. Inform us and we will send a gift.