Anti-STMN2 antibody (ab21190)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 82090 DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE protein extract PRIMARY ANTIBODY STMN2 1:1000, then 1:2000 DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED used another antibody to the same gel, which was 150 kDa, and membrane was cutted for both of antibodies ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION Ripa buffer+ PMSF (protease inhibitor) AMOUNT OF PROTEIN LOADED 35, 70, 140, 280 ELECTROPHORESIS/GEL CONDITIONS resolving gel 8% TRANSFER AND BLOCKING CONDITIONS transfer to nitrocellusoe mambrane 250mA 1 hour, blocking with resistant milk 1% SECONDARY ANTIBODY Enco Peroxidase-conjugated AffiniPure Rabbit Anti-Goat?? IgG (H+L):10000 dilution, 1 hour incubation at room temperature foolowed three washes each of 10 minutes by TBST HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Second time I took more protein and used higher concentration of primary antibody

ANSWER:

I'm sorry to hear you are having a problem with ab21190.

This antibody has been tested in human brain lysate lysed in RIPA buffer containing protease inhibitors. You do not mention which type of cell lysate you have used and it may be that your samples express low levels of the protein. I would therefore like to recommend to run the positive control of human brain lysate. I would also like to recommend to use other protease inhibitors in your lysis buffer as PMSF is not sufficient alone to prevent proteolytic degradation of proteins.

We typically recommend the following inhibitors (or ready to use cocktails which can be purchased through Roche or Sigma for example): Aprotinin 2 µg/ml,Leupeptin 5-10 µg/ml, Antipain 2-10 µg/ml, Pepstatin A 1 µg/ml, Na-Fluoride 5-10 mM , Orthovanadate 1 mM, PMSF 1 mM.

Secondly the problem may be due to low binding of the antibodies. You do not mention how long you incubate the primary antibody for; I would like to recommend to incubate overnight at 4C, and to try more concentrated antibody (try 1ug/ml). The problem may be also partly due to low blocking. We typically recommend to try 5% BSA and also to try 5% milk for 1 hour at room temperature. Then incubating the primary and secondary antibodies in TBST only (0.1%Tween20). Can I finally please make sure that the conditions you use are denaturing and reducing (i.e. the samples are boiled in loading buffer and run on a reducing gel?) and that the secondary has been tested with other primary antibodies and therefore is known to be working.

Please let me know if this helps and do not hesitate to contact us for further advice.

Hi,

A customer ordered ab21190 about two weeks ago and tried it in three different WB without success. The conditions of the assay were as follows:

SDS PAGE gel 8% or 15% acrylamide Sample: 300 ug mouse brain lysate Primary ab: 7.5 ug (15 ul from the AB solution) in 15 ml (=1:1000) or 5 ug (10 ul) 5 ml (1:500). Secondary ab: HRP conjugated donkey anti-goat (Jackson Immunoresearch lab) (1:10000) acoording to manufacturer Detection: LumiGLO reagent and Peroxide (Cell Signalling) according to manufacturer

In the same WB different antibodies (all of them to AChE) recognized AChE in the homogenate. I.e., the experiment itself did work.

Do you have sggestions, or should we offer a replacement?

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. I am most surprised by the results that your customer has been obtaining. We recommend that the antibody can be applied in a dilution range of 1:2000 to 1:5000. Your customer has been applying the antibody at a significantly higher concentration than we recommend and it is confusing to me why they have not obtained better results.

We recommend that brain lysate is applied as a positive control as your customer has been using. However, my concern is that they are loading 300ug of protein; something that I would not recommend. We recommend that 20ug of lysate is loaded. Please can you clarify this. I am also interested in the method of lysate preparation and the results that your customer has obtained using their AChE antibody. I would like to recommend that a RIPA buffer extraction is performed and 20-30ug is loaded onto the gel.

I am in touch with the originator of this antibody to determine the conditions employed as I am concerned about the results that your customer has obtained using their positive control lysate. In the mean time I would appreciate comments on the questions above. I look forward to hearing from you.

BATCH NUMBER 150158 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No signal or weak signal. I have tried this antibody three times and each time I have increased the primary antibody dilution (1:2000, 1:1000, 1:500). With the 1:500 dilution I got a very weak doublet around 30Kda after exposing for 15min (ECL detection system). I also varied the secondary dilution, however, it has not really had an effect on the signal.

SAMPLE rat cell extract

PRIMARY ANTIBODY abcam/goat/5%milk TBS/1:2000,1:1000,1:500/2 hour incubation, 5 quick rinses in DI water, 10 min TTBS

DETECTION METHOD ECL

ANTIBODY STORAGE CONDITIONS 4 degrees

SAMPLE PREPARATION tris HCL buffer (pH 8), Protease Inhibitors (Benzamidine, Okadaic Acid, Cypermethrin, Leypeptin, Orthovanadate, EDTA, PMSF). Sample heated for 10min at 70 degrees.

AMOUNT OF PROTEIN LOADED 50ug

ELECTROPHORESIS/GEL CONDITIONS Non reducing, 10% bis-tris

TRANSFER AND BLOCKING CONDITIONS 15% methanol NuPAGE Transfer Buffer, Blocking in 5% Milk/ TBS solution

SECONDARY ANTIBODY [competitor]/rabbit anti goat/5% milk TBS/1:2000/2 Hours/ 5 quick rinse, 10 min TTBS

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? varied primary and secondary dilutions

ANSWER:

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab21190. Please note that this antibody is a "Fast-track" antibody and has not yet been fully characterized. Preliminary Western blot experiments were done using human brain lysate and an approx 24 kDa band was detected. Please note that currently we cannot find an explanation in the literature for the band we observe given the calculated size of 20.8 kDa.

I'm not sure exactly what type of rat cell extract you are using, but you may want to try using a human brain lysate to compare your results to. I would also suggest extending the incubation period with the primary to overnight at 4C. Also, I noticed that you used non-reducing Western blot conditions; this antibody was tested using reducing conditions, so please try that.

As this is a fast-track antibody, we cannot guarantee that the antibody will work in any application other than ELISA against the immunizing peptide and therefore we are unable to offer a refund if the antibody does not work in your application. However, we would greatly appreciate your feedback on these antibodies, whether positive or negative. We will award 1,000 Abpoints to the first researcher who sends us positive feedback (including an image and details of materials & methods). Conclusive negative feedback that leads us to withdraw the antibody will also be rewarded with 1,000 Abpoints. Other useful positive and negative feedback will be rewarded with 50 Abpoints. All awards are subject to approval by Abcam scientists and provision of suitable data. Please submit feedback to fast-track@abcam.com