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Frequently Asked Questions (FAQs):
Q: How should I store my tube of antibodies? How much does temperature affect stability?
A: Please store the antibodies in the original tube at -20°C and do not aliquot. Avoid repeated freeze!
Q: How should I store my SDS samples of phosphorylated proteins?
A: SDS sample cell lysates can be stored at -20°C for short-term use (less than 3 months), but should be kept at -80°C for long-term storage. The degradation of activated protein samples varies greatly, depending upon the nature of each protein and how much activated protein is within the samples.
Q: Can I incubate this antibody for 1 hour at room temperature instead of overnight at 4°C?
A: Our activation-specific antibodies are purified to distinguish between only one or two post-translationally modified amino acid residues. These modifications include phosphorylation, acetylation and enzymatic cleavage. Most antibodies raised against such modified sites have a lower affinity when compared with antibodies recognizing whole proteins. Thus, it is critical to prolong antibody and antigen interaction time to obtain best signal to noise ratios by incubating overnight at 4°C.
Q: How do I prepare good control cell lysates for this phosphorylated target protein?
A: Please refer to the Western blot figure on the data sheet for the product that will demonstrate a suitable control cell line and drug treatment.
Q: If a species is not listed as cross-reactive with an antibody on the data sheet, does this mean that the species has not been tested?
A: If we do not obtain positive results in one species, we do not list that species. However, it is possible that the cell line we test may contain a low level of particular protein or the inductions we used do not work in that particular cell line. Therefore, we highly suggest you compare the peptide sequence around the modification site (5 amino acids on each side) between the species you are studying and the species that our antibody was raised against. In most cases, our antibodies are raised against the human protein sequence and are highly purified using affinity chromatography. Many times if the sequences are identical or have only 1 or 2 amino acid differences in residues other than the phosphorylation site, there is a good chance the antibody will recognize your protein.
Q: Why can't I use my own lab's Western blotting protocol with your antibodies?
A: Our antibodies are optimized to provide the strongest signal with the least background, using our standard Western blotting protocol. The benefit to the end user is the saving of costly time and materials optimizing to your own procedures.
Q: Why isn't this antibody working by Western blot?
A: There are several details of our Western blotting procedure that are critical to avoid loss of signal and or high background. Do not over wash the membrane. Washing should be performed with TBS/0.1%Tween-20 three times for 5 minutes each. Excessive washing can wash away the primary antibody. The recommended washing conditions are optimized to generate the best signal to noise ratio and therefore the strongest, cleanest results. Do not over block (e.g., for more than one hour or overnight). Excess blocking can reduce the specific signal seen with some antibodies. Be sure to consult the individual product's data sheet for the recommended blocking time for each antibody. Is the species you are testing listed in the cross-reactivity section of the datasheet for the antibody? Did you load at least 20 µg of total cell lysate? Did you induce the activation of the protein in your cell lysate samples?
Q: How can I ensure reproducible Western results?
A : There are several possibilities as to why Western-blotting results may seem difficult to repeat. To discover the cause of this situation, consider the following: How did you store the antibody? The antibodies cannot be re-used after dilution in the primary dilution buffer. Many antibodies have a very low working concentration and it is possible that the concentration will decrease with each successive blot. Are you certain that your samples contain the activated protein? Cell lysate sample preps for activated proteins can vary due to cell number, drug induction timing, cell passage number and storage conditions. Our products can be expected to perform at optimal levels during their shelf-life period. Please note: Do not aliquot the antibodies.
Q: How do I use immobilized antibodies for immune precipitation?
A: It is important to obtain homogeneous bead slurry for optimum results. The following steps will ensure such homogeneity: Place the tube containing the bead slurry in an ice bucket for 10 minutes. Invert the tube several times to attain a 50% bead-to-buffer slurry. If necessary, gently vortex the tube. To pipette the product accurately, cut the end of a pipette tip off and insert the tip into the bead slurry just enough to draw up the immobilized antibody bead mixture. Incubate the immobilized antibodies with 200 µg of total cell lysate (approximately 1 mg/ml) with the recommended antibody dilution overnight at 4°C.
Q: Does this antibody work for immunohistochemistry?
A: We routinely tests antibodies for use with formalin-fixed paraffin-embedded samples. Those that pass rigorous validation standards are recommended for IHC-P. We also test products on fresh frozen tissue samples, and those that stain successfully are recommended for IHC-F. Those products that do not have IHC-P or IHC-F listed as a recommended application may have failed, may not have been tested, or may require specific tissue samples to which we do not have access. It is suggested that you contact us regarding the nature of a lack of an IHC recommendation for your product of interest, as there may be data that suggests it could work well in another system. Alternatively, we may have data that suggests another product would be a better choice for tissue staining.
Q: Does this antibody work for immunofluorescence?
A: We have tested all antibodies in immunocytochemistry using the ABC method in most cases and by immunofluorescence in some cases. The end results are shown in the data sheet and on the website, or contact us atsupport@dijibio.com for further details.
Q: Which antibodies are optimal for specific applications?
A: Please refer to our product specific comparison tables to determine which antibody works best for you.
Q: How do you ensure the quality of antibodies?
A: We strive to test all our products on major applications such as Western Blot, immunocytochemistry, immunohistochemistry and ELISA. We routinely test our antibodies on different species including human, mouse and rat. The high quality of our products is the most important consideration at DJBio. We are constantly testing our products for new applications and if you are interested in a particular application not listed on the data sheet, please contact our technical support at support@dijibio.com for the latest application update or for help with any questions you have.
Q: How do you quality control for different lots?
A: We strive to achieve product consistency or improvement from lot to lot. As a standard, we test the new lot in all the applications we recommend side by side with the old lot. The recommended dilution for each application is optimized for each lot following our protocols. Concentrations between lots of antibodies are sometimes adjusted to obtain better results.
Q: Can I order this antibody without BSA or glycerol or could you develop an antibody for me?
A: For bulk orders, many of our antibodies can be prepared to the customer's specifications. We may consider developing an antibody against a novel activation site if there is enough interest in the scientific community. For pricing and availability, please send your suggestion to order@dijibio.com and we will contact you surely and shortly.
Q: Need further help?
A: For specific technical questions and substantially expanded services , contact us at support@dijibio.com.
