Anti-hnRNP A2B1 antibody [DP3B3] (ab6102)

The order number is 15032.1.32-ET-MG.

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused.

As requested, I have issued a free of charge replacement with the order number 979027 with ab31645.

To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee.

Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.  

I would prefer a different antibody than a vial from the same (since it is monoclonal, i'm not sure if the results would improve that much). Since the Anti-hnRNP A2B1 antibody (ab31645) works for ICC/IF it might work either with IP experiments. Being so, i would be appreciated if you could dispatch the one that i've refered. Thank you for your support,

ANSWER:

For dispatching the requested product, we would need the order number of original purchase. Could you please send this asap?

Looking forward to hearing from you soon.  

What type of beads were used for IP? *Protein G-agarose beads* > How the IP was performed? Have you mixed antibody beads and lysates once, or beads and antibody first then lysates or antibody lysates and then beads? *Antibody (6ug) with 100ul of beads o.n., 4ºc in the next day, 1mg of protein was incubated during 1h at 4ºC* >For how long the mixture was incubated? *1h* >Can we define the problem as no bands in IP or multiple bands because loading 1000 ug protein per well in WB is way too high. If it is not true then I am presuming that this is the amount of protein (lysates) you have used to mix with antibody and beads. *1mg was used to the IP for the input, 50ug of protein* > 1/1000 dilution means the antibody was used at 1ug/ml which is very low I would recommend using ab at 5 ug/ml and 10ug/ml. *1/1000 was the dilution used in the 3 abreviews that were availabe together with the datasheet. So the dilution is not the problem at least in the input lane it was supposed to see a stronger band at 37kDa and none at 50kDa.* > Could you send us an image? *Sorry, i attached a file previously, but then the page have reloaded because i've spent more than 20min filling the form. How can i send you the image? I've tried to reply by e-mail but it was not delivered...

Here's a link with the image that i wanted to attach: http://imageshack.us/f/31/hnrnpa2.png/

ANSWER:

Thank you very much for sending the image and all the details.

I acknowledge the time you have spent trying this antibody and convinced that the observed band size is not at 37 kDa. The antibody should be detecting the band at right size; the rat protein is approx 99% similar to human protein. I unfortunately can‘t compare the results with our own lab data because ab6102 was never tested with rat lysates. It may be worth going through the literature and comparing the results with other scientist’s findings.

In order to proceed further in this case I can either send you a free of charge replacement of ab6102 from different lot or can also dispatch a different antibody e.g. ab31645 or ab24409 or ab64800; although these antibodies are not tested in IP however I don’t see a reason why these antibodies can’t be used in IP. If you are interested in trying these let me know I will then send you a vial of ab. 

I also want to point out the background in IgG IP which kind of show similar pattern as IP of A2; I am not sure why this is, the IgG IP should be clean; one reason could be the high protein conc. as compare to the antibody.

I will look forward to receiving your reply soon. Please do not hesitate to contact me if you have any question.  

LOT NUMBER GR21753-2 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Non-specific band WB from IP samples show no band for 37kDa zone also, the input lane shows two bands (strong one at 50kDa and a weak band at 37kDa) SAMPLE Rat primary hippocampal cell culture PRIMARY ANTIBODY 1:1000 diluted in 0,5% milk TBS-T Washed 3x 10min with TBS-T DETECTION METHOD ECF, 5min max ANTIBODY STORAGE CONDITIONS 4ºC (short-term) -20ºC (aliquots) SAMPLE PREPARATION RIPA supplemented with proteases inhibitors (CLAP and PMSF), phoshpatase inhibitor (NaF), DTT and RNase inhibitor Samples from IP were dissolved with sample buffer 2x and heated at 95ºC for 5min AMOUNT OF PROTEIN LOADED 50ug for Input 1000ug for IP ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE, tris-bicine, 12,5% polyacrylamide TRANSFER AND BLOCKING CONDITIONS CAPS-methanol, 40V o.n. 4ºC blocked with 5% milk TBS-Tween SECONDARY ANTIBODY Anti-Mouse 1:20000 diluted in 0,5% milk TBS-T Washed 3x 10min with TBS-T HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Adding the flow-through samples to the WB

ANSWER:

Thank you for your enquiry regarding ab6102 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

Band at 37 kDa in input sample indicate that the antibody is working fine and further optimizations in antibody dilutions could help getting clean band at right size.

Based on the information provided I presume that antibody is unable to IP the protein. So in order to understand the problem I would like to ask few questions; - What type of beads were used for IP? e.g. protein A or G - How the IP was performed? Have you mixed antibody beads and lysates once, or beads and antibody first then lysates or antibody lysates and then beads? -  For how long the mixture was incubated? - Can we define the problem as no bands in IP or multiple bands; because loading 1000 ug protein per well in WB is way too high. If it is not true then I am presuming that this is the amount of protein (lysates) you have used to mix with antibody and beads. - 1/1000 dilution means the antibody was used at 1ug/ml which is very low; I would recommend using ab at 5 ug/ml and 10ug/ml. - Could you send us an image?

I hope the suggestion of conc. will help to improve the results. I will also look forward receiving reply to my questions soon.