Anti-eIF4E (phospho S209) antibody (ab4774)

What is the concentration of batch 132987?

ANSWER:

Thank you for your enquiry.

The concentration is 0.5 mg/mL.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Here are two blots, one from abCAM and one from cell signaling! And all the details you requested! Lot Number 63956, Abcam order number: 49277, P.O. number: 3P668468.

1. Please describe the problem (high background, wrong band size, more bands, no band etc). Wrong size approximately 36kD and 65kD!

2. On what material are you testing the antibody in WB? Species? Brain tissue lyaste from Guinea Pig along with Pos and Neg cell line lyastae! Cell extract/ Nuclear extract? Whole cell lysate. Purified protein? No Recombinant protein? No

3. How much protein did you load? From 10 to 50 ug, we always run a standard curve with increasing amount of protein to evaluate an antibody of interest before doing the various experimental conditions. How did you prepare the lysate for the analysis (protease inhibito 50 mM Tris-HCl, Standard lysis buffer with protease inhibitors and phosphatase inhibitors. Did you heat the samples? Yes

4. Primary Antibody Specification (in which species was it raised against)? In Rabbit. ab4744, you have all the details! At what dilution(s) have you tested this antibody? 1ug/ml Incubation time, wash step? 2 hours in 1% non-fat dry milk solution in 20 mM TBST, ph 7.6 as suggested in the protocol supplied with the antibody. Three wash steps at room temp for 5 min each.

5. Secondary Antibody Specification (in which species was it raised against)? Goat. Anti-rabbit IgG whole molecule, HRP conjugate At what dilution(s) have you tested this antibody? 1:2500 Incubation time, wash step? 1 hr at room temp, wash step as for primary antibody. Do you know whether the problems you are experiencing come from th No

6. What detection method are you using? Chemiluminescent detection method.

7. Background bands Have you eliminated the possibility that any background bands coul Is the blocking step sufficient? (We recommend blocking the membra YES Are your washing steps sufficiently stringent? (Multiple short was YES At what size are the bands migrating? Could they be degradation pr Approx 36kD and 65kD Please provide an image of your blot (as an e-mail attachment, a f Attached.

8. Optimization attempts How many times have you tried the Western? Two times! And we do western almost everyday in our lab, check the band with your competitor on the image labeled Cell Signaling Technol on the right hand side.. Do you obtain the same results every time e.g. are background band YES What steps have you altered? YES, overnight incubation with primary antibody!

9. Did you apply positive and negative controls along with the samples? Please specify. YES, see band at 36 kD and not at approx. 25kD with abCAM. On the contrary, see band at lower than 29kD with cell signaling tehnol. product. Using abCAM antibody, we did not get band on western blot at the right molecular weight. Please do the needful.

ANSWER:

Thank you very much for the details that you have provided, and I'm sorry to hear that you are experiencing difficulty with this antibody. As stated on the online datasheet, this particular antibody has been characterized using HeLa cell lysates and has not yet been tested on other species although it is expected to cross-react with a wide range of other species, due to sequence homology. We have not had any other complaints regarding this antibody; the problem may very well be due to the batch you received. I can offer you a replacement batch to try or a refund, please let me know which one you would prefer.