Anti-GAPDH antibody [mAbcam 9484] (HRP) - Loading Control (ab9482)

Abcam product code ab9485

2) Abcam order reference number or product batch number GR64234-1
3) Description of the problem: Tried dilutions 1:2500, 1: 1250 and 1:625 (blocking either with 5% Milk or 5% BSA). Even at 1:625 the signal is still too weak and unspecific bands appear.
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): Crude brain lysate
Lysis buffer: 1x Laemmli buffer
Protease inhibitors: none
Phosphatase inhibitors: none
Reducing agent: beta-Mercaptoethanol contained in Laemmli Buffer
Boiling for ≥5 min? yes
Protein loaded ug/lane or cells/lane not known
Positive control: brain lysate itself
Negative control: none
5) Percentage of gel: 4-20% TGX gradient Gel
Type of membrane: PVDF
Protein transfer verified: yes
Blocking agent and concentration (5% milk or 5% BSA in TBST)
Blocking time: 1h
Blocking temperature: RT
6) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution: 1:2500, 1: 1250, 1:625
Diluent buffer: Milk or BSA
Incubation time: o/n
Incubation temperature: 4°C
7) Secondary antibody: Cy3 goat anti-rabbit IgG (H+L)
Species: goat
Reacts against: rabbit
Concentration or dilution: 1:2500 or 1: 1250
Diluent buffer: Milk or BSA
Incubation time: 1h
Incubation temperature: RT
Fluorochrome or enzyme conjugate: Cy3
8) Washing after primary and secondary antibodies:
Buffer: TBST
Number of washes: 5x 5 min
9)Detection method: Imaging system with CCD camera
10) How many times have you run this staining? 4 times
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? Primary antibody concentration and blocking medium, dilution of 2ndary AB
Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

ANSWER:

Vielen Dank für Ihre Antwort und für diese weiteren Informationen.


In der Tat sieht es so aus, als ob der Antikörper unter diesen Bedingungen funktioniert haben müsste. Zwar bekommen Sie ein Signal, aber es ist in der Tat viel zu schwach bei dieser hohen Konzentration (auch wenn man schön den Unterschied zwischen Milch und BSA sehen kann).


Ich weiß, dass Sie viel Zeit für diese Experimente verwendet haben und möchte Ihnen daher einen Ersatz oder einen Gutschein als Ersatz anbieten (vorausgesetzt, das Produkt wurde in den letzten 180 Tagen gekauft). Dafür benötige ich jedoch ihre ursprüngliche Bestellnummer oder einen Kontakt in Ihrer Einkaufsabteilung und das Datum der Bestellung. Würden Sie mir bitte zusammen mit diesen Informationen mitteilen, welche Alternative Sie bevorzugen?

Vielen Dank für Ihre Kooperation und Hilfsbereitschaft. Ich freue mich bald wieder von Ihnen zu hören.

WB with mouse tissue: muscle, heart, brain, liver, salivary gland
is GAPDH expressed in these tissues?
send database entries

ANSWER:

Thank you for contacting us.

The database entries (UniProt and Human Protein Atlas) for the human and mouse GAPDH proteins are:

Human Protein Atlas: http://www.proteinatlas.org/ENSG00000111640

UniProt Database for mouse: http://www.uniprot.org/uniprot/P16858
UniProt Database for human: http://www.uniprot.org/uniprot/P04406

Especially, the Human Protein Atlas will give you more information about tissue expression for thehuman protein. I would assume one could extrapolate this also to mouse tissues. But you can always check also the literature (PubMed) for publications in that regard.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thanks for the generous offer. It was for two antibodies including two vials of Ab21873. Note that the order dates from 28/9/2010 and as I pointed out in my previous E-mail, we did receive replacements with an older batch. All I can say is that if lot number GR21097-1 works for chromatin immunoprecipitation, we will be happy to pay for it.

ANSWER:

I have issued a free of charge replacement with the order number 1042072.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that typically our Abpromise guarantee holds for 6 months from when you receive the antibody,although I have made an exception in this case.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.

Thank you for the offer of a replacement vial. However, I suspect that the previous "complaint" you refer to came from us and Abcam kindly replaced the antibody with an earlier batch. I don't think we should expect more.



My question, which was simply prompted by an E-mail from Abcam, was whether there is now a more recent batch of ab21873 (presumably lot GR21097-1) and if so, would it be possible for us to test an aliquot in ChIP conditions before buying several vials.

ANSWER:

Thanks for your reply.

You are correct that lot GR21097-1 is a new lot we have in stock. We are not able to stock small sample aliqots of our antibodies unfortunately. However, since a vial of ab21873 you previously purchased is not working as expected,I can provide you with a free replacement vial of this new lot based on our Abpromise guarantee http://www.abcam.com/abpromise

If you would like to go ahead with the replacement offer, please just provide me with the PO number or Abcam order reference number associated with the purchase of ab21873, lot 937205 so I can process the replacement.

As it happens, we have been having some issues with batch-to-batch
variation with an antibody against CBX7 (AB21873) that we use extensively
for chromatin immunoprecipitation (ChIP). Lot # 937204 worked very well
whereas Lot #937205 did not. We did manage to get what we believe may
have been the last aliquots of the good batch but are just about to run
out. If there have been any more recent batches, we would be very keen to
try some before restocking.

ANSWER:

Thank you for your enquiry. I am sorry to hear of the trouble you are having with lot 937205 in ChIP. I looked in our records and there was one other complaint of a similar nature regarding ChIP with this lot of ab21873. We do have a new lot in stock now (lot GR21097-1) and I am happy to offer a replacement for the faulty vial of ab21873.

Please pass along the PO number or Abcam order reference number associated with the purchase of ab21873, lot 937205 so I can process the replacement.