Anti-Stathmin 1 antibody (ab11269)

Dear sir : Our customer has the problem with the antibody ab11269 in IHC experiment,please check the protocol . 1. Order details: a.. Batch number: lot:309430 b.. Abcam order or Purchase order number: ab11269-100 c.. Antibody storage conditions (temperature/reconstitution etc) : -29℃ 2. Please describe the problem (high background, no staining etc). no staining

3. On what material are you testing the antibody in IHC · Species:human · Cell Iine or tissue: tissue

4. How did you fix the samples? formalin a.. Ethanol, methanol: (was it ice cold) b.. Acetone: (was it ice cold) c.. Paraformaldehyde (percentage, perfusion fixed on not): d.. Fixation time:one night e.. Fixation temperature: room temperature 5. For formalin-fixed paraffin embedded tissue: did you apply antigen retrieval step? a.. Enzymatic method: b.. Heat mediated technique: c.. Other :H2O2 6. Blocking steps: a.. For HRP detection method: did you block endogenous peroxidases:yes b.. How did you block the unspecific binding sites: serum, BSA, Milk, other FBS c.. For how long.1 hr 7. Primary antibody a.. Specification (in which species was it raised against): · At what dilution(s) have you tested this antibody: 500X, 1000X, 2000X · What dilution buffer was used: FBS · Incubation time: overnight · Incubation temperature: room temperature.4℃ · What washing steps were done: 10 min by PBS, and repeated three more times.

8. Secondary antibody a.. Specification (in which species was it raised against): · What dilution buffer was used: no dilution · Incubation time: 30 mins · Incubation temperature: room temperature · What washing steps were done: five times · Do you know whether the problems you are experiencing come from the secondary? The secondary antibody does not change. The primary antibody that I bought last time can work，but the new one can not.

9. What detection method are you using? DAB

10. Background staining · Please provide an image of your staining

11. Which detection system did you use? DAB

12. Did you apply positive and negative controls along with the samples? yes

13. Optimization attempts · How many times have you tried the IHC?Twice · Do you obtain the same results every time? Yes.the result is not good。 · What steps have you altered? no

ANSWER:

Thank you for contacting us on behalf of your customer.

The customer mentions that a previous vial of the antibody work, if the customer is using the same protocol as before this would indicate that the current vial is damaged and I would be happy to consider giving a replacement vial to the customer or a refund if the recent vial was purchased in the last 120 days.

I would appreciate if you can provide the lot number that worked for the customer, so I can see if it was from the same master stock.

Please also provide your order details to arrange for the replacement.

Thank you for your suport,

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 00019527

DESCRIPTION OF THE PROBLEM Multiple bands stathmin is a phosphoprotein. Its predicted size is 18 kda. I do see this band clearly, but I do see a band directly above it. Does this antibody also recognize the phosphorylated form of stathmin? That is does this antibody recognized total op18?

ANTIBODY STORAGE CONDITIONS -20

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

Thank you for your enquiry. Your enquiry has been passed on to my from my colleague Cindy.

Following a brief literature search I have determined that Stathmin (Phosphoprotein p19) is phosphorylated at Serine-26 and Serine-38. Please see the publication below. This antibody does not distinguish between these residues as the immunogen sequence used to raise this antibody was a synthetic peptide, corresponding to C terminal amino acids 132-149 of Human Stathmin 1. Therefore I consider it highly likely that this antibody is detecting the non-phosphorylated and phosphorylated forms of Stathmin 1.

Analysis of phosphoprotein p19 by liquid chromatography/mass spectrometry. Identification of two proline-directed serine phosphorylation sites and a blocked amino terminus.Labdon JE, Nieves E, Schubart UK. J Biol Chem. 1992 Feb 15;267(5):3506-13.

http://www.jbc.org/cgi/reprint/267/5/3506

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

what is the protein concentration of this product??

ANSWER:

Thank you for your enquiry. The concentration for the lot that is currently in stock is 4.5 mg/ml.

Please contact us again if you have any additional questions.