Anti-TNF alpha antibody [P/T2] (ab9579)

Thank you for your enquiry. I have attached a copy of the IHC image that is on the datasheet for the antibody, a granulomatous inflammatory reaction, probably in a chronic cholecystitis. The species is human. Please let me know if you have any questions.

Thank you for your inquiry.

I am sorry to hear that your customer is having trouble with these antibodies.

It seems to me that there must be an issue with the HRP polymer detection system, DAB,or antigen retrieval buffers/system. The reason is that Iooked up the previous Sellex orders for this product and the customer is comparing the same lots with the old and new batches for two products.

For ab49679, CD105, the customer is having trouble with lot GR41983-2. The previous lot that worked for them on the earlier Sellex order was GR41983-1. These are the same lots, except the -2 was aliquoted after the -1.

For ab66184, VEGFR1, the customer is having trouble with lot GR30735-3. The previous lot that worked for them on the earlier Sellex order was GR30735-2. Again, these are the same lots, except the -3 was aliquoted after the -2.

For ab74525, TGF beta-1, the cutomer was having trouble with lot GR69795-1. This lot is different than the GR14885-2 lot that worked well for them on the earlier Sellex order. So there may be a lot variation in this instance, but I would recommend using all new buffers in the protocol to ensure this.

Both ab9542 and ab9579 were purchased on the same order back in June 2011, which is outside our 6 month Abpromise period.

There must be something going on with the customer's detection system, buffers, or perhaps they are using a frost-free freezer, or thefreezers went down,etc. It wouldn't make sense for all 5 antibodies to show decreasing signal, requiring more antibody. Especially because they are using 2 of the same lots for 2 antibodies, there can't be lot-to-lot variations in these cases.

I hope this information helps. Please contact us with any other questions.

Thank you for contacting us. I have received the following information from the lab: The antigen retrieval performed with this antibody in paraffin sections is: 5 minutes heat-induced epitope retrieval (HIER) in boiling citrate buffer, pH 6.0. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your enquiry. From the three antibodies that you list I would like to recommend TNF alpha antibody (ab6671). This antibody has received a favourable Abreview from one of our customers detailing its application following citrate antigen retrieval. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Thank you for your enquiry. The best positive control for this antibody is human macrophages in a granulomatous inflammatory lesion. I hope this information helps. Please do not hesitate to contact us if you need anything further.

Thank you for your enquiry. I am sorry to hear that you have been experiencing problems with ab8999. As discussed on the telephone this afternoon please would you fill in the questionnaire by clicking on the link below. I would also appreciate it if you could send me details of the LOT number and images of the blots for both ab8999 and the anti-Prp (ab3408) that we discussed. This will give me an appreciation of what you have done to optimise this antibody by western blot. I hope that we can resolve this problem rapidly. https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=8999&mode=questionaire I look forward to hearing from you.

Thank you for your email. Did you order the antibody directly from Abcam or from one a distributor, such as Biozol Diagnostica? If you ordered from a distributor, please indicate which one and also the purchase order number that you used with them. The replacement will then be shipped to you via the distributor. I also received the following information regarding the Western blot protocol that was used when the antibody was tested which I wanted to pass on to you: "Sample preparation 0.5% Triton-X100 containing PBS with protease inhibitors (1mMPMSF, 1ug/ml aprotinin and leupeptin) 45 min on ice. Sample buffer for SDS-ELFO Boiling for 5 min in 0.5M Tris-HCl buffer contain 10% SDS and 1% mercaptoethanol, 25% glycerol ELFO Separation on 10% polyacrylamide gels according to Laemmli (Running with 200V). Immunoblot Blotting to nitrocellulose sheets (Schleicher & Schuell) overnight at 35mA. Blocking with 5% non-fat milk powder for 1 hour then incubation with anti-TNF-? mAbs (1-5 ug/ml) for 1 hr, then HRP conjugated anti-mouse antibodies used as second reagent (Dako) for 1 hr. Development with ECL reagents (Amersham). PBS containing 0.05% Tween-20 for washing among the reaction steps."

Thank you for your message. I have checked with the originator of this antibody and there have not been any complaints concerning this lot. I'm still waiting to obtain some more information regarding the use of this antibody in Western blotting, but I can send you a replacement lot to try if you wish. Please let me know and also when you would like it shipped as many places will be closed over the holidays. Thank you, and happy holidays!

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with this antibody. I would like to make a few suggestions to try to help you out. To see if you can obtain a signal, try incubating with the primary antibody overnight at 4C. Make sure that the protein transferred properly to the membrane (check this by using Ponceaue S). Also, increase the concentration of the secondary antibody. Is the secondary antibody working with other primaries? You may also want to decrease your number of washes. If you still are not seeing a signal, please do let me know.

Yes, ab9570 will work using Envision + mouse and/or Envision mouse-rabbit kit.