Anti-YB1 antibody (ab12148)

First of all, thank you for all of your thorough help. I too have noticed
from the literature that depending on cell type, YBX1 can show up in
either nucleus or cytoplasm, so my initial nuclear results were not
entirely unexpected. Though neither would cytoplasmic staining have been
surprising.
If you have another lot for me to try, that would be great. My greatest
regret is that I don't have any of the original lot left to test with a
blocking peptide. As it is, my greatest hope I suppose is to try a new
lot and hope to replicate my initial results.
The address (which I'm sure you have from previous orders) is:

Thanks again for your help.

ANSWER:

Thank you for confirming that.
I have now arranged for the new vial to be sent out to you. The new order number is 1074326 (purchase order FOCR 56072). Please let me know if you have any problems receiving this.
I hope this lot achieves the results you are hoping for. Please do let me know how you get on.
Until then, I wish you all the best with your research.

Thank you for all of your help with this, and I'm sorry it's not more
straight-forward.
I'm not sure I can find the order number for the old YB1 antibody lot. I
have the data sheet though and it says 'printed on 14 June, 2006' which is
around the time I ordered it. It was delivered to
The data sheet for the newest lot says 'printed on 18 June, 2012'. It was
delivered to the same address.
I have ordered this same antibody at least one additional time between
these two times, but have used it in other applications and as a result
cannot know if it functioned in immunohistochemistry like the 2006 lot or
the 2012 lot.
I hope this helps, if you need more information, I'll talk to our lab
manager about the possibility of a distributor or if I can track down an
order number. Thanks.

ANSWER:

I am sorry for the delay in getting back to you in respect to your report of obtaining varying staining using ab12148.
The lab has confirmed that looking back at the production notes for this antibody, there has been no difference in any purification or production process which would explain the difference in staining. We only test this antibody in house in IF so it has not been batch tested in IHC before going into stock.
I've done a quick literature search and it seems that YB1 can be detected in the nucleus and cytoplasm depending on the stage of the cell cycle but should be largely cytoplasmic, as is the case with the newer lot number you used. This has also been observed through staining of HeLa cells presented on the datasheet of ab12148.
It may be that the older lot unfortunately displayed some non-specific staining when using IHC which was not picked up in the quality control process at that time. If you would like, I can send an alternative lot of ab12148 for you to try to see if you obtain the same staining again, or if you would prefer, I can send the blocking peptide (ab12411) which can be used to observe the specific staining. Unfortunately, we do not have this blocking peptide in store currently so this would take 4-6 weeks to be delivered.
I hope this information has been of some help. If you have any further questions, please do not hesitate to ask.

Good afternoon,
I am a graduate student researcher and have a question regarding a rabbit
polyclonal Ab to YB1 (ab12148).
I originally got this product in the summer of 2006, and found it to work
beautifully in fluorescent immunohistochemistry. I recently purchased it
again in Feb of this year, and in repeating the experiment found a very
different result. The staining still worked (in comparison to isotype
control), but the staining was positive in different cells in the tissue,
and different intracellular location was observed. The tissue was of same
mouse strain/age/gender etc, and the conditions (secondary, antigen
retrieval etc) also were also unchanged.
I presume I am not working with the same lot (with such a great amout of
time passing between the two purchases), but I was wondering if you know
of any problems with this product, dramatic changes in its production or
what might have caused this variation from one result to the next.
Thanks for your help.

ANSWER:

Thank you for contacting us. I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.
You are correct in thinking it is most likely a new batch of antibody you used now in comparison to that used in 2006. I have looked into the antibody history and it does not seem that we have had any batch related problems reported with this antibody and our QC data does not indicate that any significant change has occurred. The antibody is raised against the same immunogen peptide and tested in the same way. I am checking with the lab to see if they can shed any further light on this issue.
In the meantime, would you mind sharing a little further information with me in regards to the experiment you have been performing and the differing results you have obtained with the antibody so that I can investigate this case further? I have attached a questionnaire to this email for this purpose. If you have the details of the lot number you have used now, as well as that of 2006 that would be of great help. If you do not have this information then the delivery address used on both occasions should be sufficient for me to track down the order details.
I look forward to receiving your reply.

The paper about YB-1 cleavage product is, "Sorokin AV, Selyutina AA, Skabkin MA, Guryanov SG, Nazimov IV, Richard C, Th'ng J, Yau J, Sorensen PH, Ovchinnikov LP, Evdokimova V. Proteasome-mediated cleavage of the Y-box-binding protein 1 is linked to DNA-damage stress response. EMBO J. 2005 Oct 19;24(20):3602-12."

I would like to ask you if this blocking peptide is the peptide that you used to generate the antibody, and when you use the blocking peptide to test the antibody, are you saturating the epitope/s with the blocking peptide and antibody is not able to bind because of competition with the blocking peptide?

It would be great if this antibody was able to detect the full length protein and the cleavage product, however, we also think that 36 kDa protein is a non-specific product. Thank you very much for your help.

ANSWER:

Thank you for getting back to me.

Yes, you are correct in your assumption. For the blocking peptide experiments the antibody is incubated with the peptide used for immunisation prior to immunoblotting. The antibody is prevented from binding to the antigen on the membrane due to competition with the peptide.

However, your siRNA experiments do indeed suggest that the 36 KDa band is in fact a cross reacting product. This is based on the assumption that the 36KDa cleavage product is indeed derived from the 50KDa product.

I am certainly prepared to offer you credit against your initial purchase provided that this antibody was purchased within the past 90 days. If this is the case please provide me with your purchase order number and date of purchase.

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

BATCH NUMBER 63000 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Antibody is detecting two bands, 36kD and 50kD, as described in the Abcam data sheet. 36kD band is the predicted band and 50kD band is a cross reacting band but it is nature is unknown. Abcam refers the paper from Evdokimova that was published in 1995. However, according to the recent literature, 50 kD (or 54kD) band is the the full length YB-1 protein (hence it is also called p50) and 36kD (or 32kD) band is the cleavage product. There is another cleavage product which is around 22kD. By using Abcam antibody, we are able to demonstrate these three bands; full-length protein and two cleavage products. Therefore, during the initial use of the YB-1 antibody we didn't encounter any problems. As there is still a controversy about the nature of the cleavage products, we knocked down full length YB-1 protein by siRNA. This is repeated two times, after the first siRNA transfection, we repeated the transfection for a second time a week later and harvested the cells another week later (day 1 first transfection, day 8 second transfection, day 15 harvest). We observed complete knock down of 50kD protein, however, 36kD product was still being observed with even more intensity. 22kD product also showed inhibition. According to your observations, 36kD band is the predicted band and we are not able to explain why siRNA had no effect on 36kD.

SAMPLE Human cancer cell line extract

PRIMARY ANTIBODY Abcam YB-1, rabbit, 5% BSA as diluent, 5000X dilution, overnight incubation at 4C, TBS-T wash 3x 15 min

DETECTION METHOD ECL detection

POSITIVE AND NEGATIVE CONTROLS USED Positive control was non-treated cell lysate.

ANTIBODY STORAGE CONDITIONS Antibody is in -20C freezer.

SAMPLE PREPARATION RIPA-B buffer containing: Roche complete mini pill, PMSF, sodium orthovanadate, sodium pyrophosphate, sodium fluoride

AMOUNT OF PROTEIN LOADED 15 micrograms

ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PAGE gel

TRANSFER AND BLOCKING CONDITIONS Towbin transfer buffer, 1 h transfer, 5% BSA as blocking agent

SECONDARY ANTIBODY Cell Signaling Technology, anti-rabbit, 5% BSA as diluent, 5000X dilution, 1 h incubation at room temperature, TBS-T wash 3x 15 min

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 30-40 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 handed to me by my colleague Tanya Barij. Thank you for your patience in this matter. I have examined your technical questionaire and I have a few comments.

The results that you have been obtaining are indeed interesting when compared to the results that we have obtained in house using the blocking peptide. The blocking peptide experiments suggest that this antibody in fact recognises the full length protein (50KDa) (p50; as you highlight) in addition to a potential cleavage product at 36KDa. However, in view of the results that you have obtained using siRNA knock down experiments it is highly likely that this product is in fact a non-specific product migrating at a similar size to a potential cleavage product.

I apologise for my lack of knowledge of this protein but I could not find reference to YB1 undergoing cleavage. I would appreciate if you could highlight some literature where this is detailed. The literature that I did find suggested that this protein is "a 36 kDa protein with anomalous electrophoretic mobility by SDS–PAGE. Please can you confirm these details as to whether this protein undergoes cleavage.

I appreciate your input with regards this. This will enable me to have a better picture of the specificity of this antibody and enable me to update the datasheet to reflect this.

I look forward to your response.