Frequently Asked Questions

1. How should I store my tube of antibodies? How much does temperature affect stability?

Please store the antibodies in the original tube at -20°C and do not aliquot. The antibodies are provided in a buffer that contains 50% glycerol, so there is no possibility of degradation of the antibody due to repeated freeze-thaw cycles.

2. How should I store my SDS samples of phosphorylated proteins?

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. It depends on the nature of each protein and how much activated protein is within the samples.

3. Can I incubate this antibody for 1 hour at room temperature instead of overnight at 4°C?

CST's activation-specific antibodies are purified to distinguish between only one or two posttranslationally 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.

4. What buffer should I use to prepare my cell lysate samples?

Please check the recommended buffer in the Western blotting protocol included with each product's data sheet. The lysis buffers are also available as products (see below), or the specific buffer components are listed on the CST website if you would like to prepare them yourself. Please note: It is absolutely critical to use CHAPS Buffer (10X) #9852 in preparing cell extracts with the antibodies for caspases and caspase substrates.

Sample	Buffer
Use the standard SDS PAGE loading buffer #7722 or #7723 for whole cell lysates.	Blue Loading Buffer Pack #7722 or Red Loading Buffer Pack #7723
Use #9803 for immunoprecipitation and/or kinase assays.	Cell Lysis Buffer (10X) #9803
Use #9852 for Western blotting with cleaved caspases and caspase substrates.	CHAPS Buffer (10X) #9852

5. Will CST antibodies work on my whole tissue sample lysates?: CST does not test all their antibodies on whole tissue lysates in-house, but many of our antibodies have been used by other customers on whole tissues with great success.
6. How do I prepare good control cell lysates for this phosphorylated target protein?: 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.
7. If a species is not listed as cross-reactive does this mean that it hasn't been tested?: CST scientists test all CST antibodies on human, mouse and rat species if possible. 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 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 the CST antibody was raised against. In most cases, CST 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 possibility the CST antibody will recognize your protein.
8. Why can't I use my own lab's Western blotting protocol with CST antibodies?: CST 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.
9. Why isn't this antibody working by Western blot?: There are several details of CST's 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.
In general, use an anti-rabbit secondary for polyclonal antibodies and an anti-mouse secondary for monoclonal antibodies. But check the data sheet available on the product pages.
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?

10. How can I ensure reproducible Western results?: 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? Please refer to the first two General FAQs.
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.
The shelflife of CST products is one year from the assay date when stored under the recommended storage conditions. Please note: Do not aliquot the antibodies.

11. How do I use immobilized antibodies for immunoprecipitation?: 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.

12. Does this antibody work for immunohistochemistry?: CST scientists have checked all antibodies recommended for IHC on paraffin sections using our paraffin protocol. However, negative results may be due to a low level of protein or modification in the particular tissue we have tested. Antibodies are not tested on cryosections at CST, but some CST antibodies have been tested successfully by collaborators or customers on cryosections. Please check the product's data sheet.
13. Does this antibody work for immunofluorescence?: CST scientists 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 check with the product manager at info@uk.neb.com for further details.
14.Which Akt antibody do you recommend works optimally for which application?: Please refer to this table in determining which Akt antibody works best for you.

15. How does CST ensure the quality of their antibodies?: CST scientists strive to test all our products on major applications such as Western immunoblotting, immunoprecipitation, 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 CST. 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 CST technical support at techsupport@uk.neb.com for the latest application update or for help with any questions you may have.
16. How does CST quality control for different lots?: CST scientists 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.
17. Can I order this antibody without BSA or glycerol?: For bulk orders, many of our antibodies can be prepared to the customer's specifications. Please contact us for pricing and availability at bulks@uk.neb.com.
18. Could CST develop an antibody for me?: CST may consider developing an antibody against a novel activation site if there is enough interest in the scientific community. Please send your suggestion to info@uk.neb.com and a CST scientist will contact you shortly.
19. Need further help?: For general product questions, contact info@cellsignal.com.
For specific technical questions, contact techsupport@uk.neb.com .