Anti-p53 antibody [PAb 240] (ab26)

Thank you very much for your prompt response and the information on the replacement antibodies! Can you please send me AB28 as it may be better suited for rat samples based on the Datasheet?

ANSWER:

Thank you very much for your reply.

I'm sending a free of charge vial of ab28 on the order ***, and this order should arrive tomorrow. Please keep me updated about the results using this replacement antibody.

If there is anything else that we can do for you, please don't hesitate to ask. I'll be happy to help.

Thank you for your email. We tried a Western using the Abcam ab26 anti-p53 antibody that I bought a while back. Because of the high concentration of antibody recommended, we piggybacked two lanes on a gel. One lane is our rat cell samples and the other is a p53 positive control from another company (treated Hela cell prep).

Basically, we loaded 30 ug protein /lane. After transfer, Ponseau S stain did show good separation and transfer. The blot was blocked for 1 hr at RT with 5% milk in PBS-0.1% tween 20. The blot was then incubated with anti-p53 ab (1:200) in 5% milk-T-PBS overnight at 4 °C. The next day, after washing, the blot was incubated with HPR-anti-mouse (1:2000, 1 hr RT) followed by ECL development. As you can see, not only we did not pick up a signal with our rat cell prep, no definitive signal is seen with the Hela cell prep which I can not explain.



During our conversation on the phone, you mentioned that Abcam has several other p53 antibodies. I wonder if we can try one of those for better detection of rat p53.

ANSWER:

Thank you for your reply and for keeping me updated. I'm sorry to hear that the results were so poor with this antibody.

I would be happy to send a replacement antibody that might work better. We do have several other p53 antibodies with rat reactivity, but I would recommend ab28 or ab80645, as these have been used in the most publications and have the most positive feedback from other users-

http://www.abcam.com/p53-antibody-P-ab28.html

http://www.abcam.com/p53-antibody-DO-1-BSA-and-Azide-free-ab80645.html

Would you like to try one of these? They are both in stock and can be shipped out promptly.

Please let me know if you have any questions or need anything else from me.

At what dilution should the antibody be used in Western blot?

ANSWER:

Thank you for your call today and for your questions about ab26.

As we discussed, I would be happy to help if you have any trouble using ab26 in your Western blots. Please keep me updated aboutyour results and let me know if you have any questions or if there is anything else that we can do for you.

Have a good evening!

I would like to know if you could provide me with references for the Anti-p53 antibody [PAb 240] (ab26) in Human Immunostaining.
Thank you.

ANSWER:

Thank you for contacting Abcam regarding ab26.


All of the available references for this antibody are listed on the online datasheet. Since I do not have access to all of these journals, I cannot say for sure which, if any, specifically refer to the use of the antibody in human immunostaining. For your convenience I have included the list of references below:


Guillen-Ahlers H et al. Fas/CD95 Deficiency in Apc Mice Increases Intestinal Tumor Burden. PLoS One 5:e9070 (2010). IHC-P; Mouse. PubMed: 20140201

Shen W et al. Female infertility in PDE3A (-/-) mice: Polo-like kinase 1 (Plk1) may be a target of Protein Kinase A (PKA) and involved in meiotic arrest of oocytes from PDE3A (-/- ) mice. Cell Cycle 9:4720-34 (2010). WB; Mouse. PubMed: 21099356

Roa W et al. Gold nanoparticle sensitize radiotherapy of prostate cancer cells by regulation of the cell cycle. Nanotechnology 20:375101 (2009). PubMed: 19706948

Pan D et al. SnoN functions as a tumour suppressor by inducing premature senescence. EMBO J : (2009). WB, ICC/IF; Mouse. PubMed: 19745809

Yeo J et al. Label-free electrochemical detection of the p53 core domain protein on its antibody immobilized electrode. Anal Chem 81:4770-7 (2009). WB. PubMed: 19435284

Izadpanah R et al. Long-term in vitro expansion alters the biology of adult mesenchymal stem cells. Cancer Res 68:4229-38 (2008). WB; Human. PubMed: 18519682

Gu BW et al. A pathogenic dyskerin mutation impairs proliferation and activates a DNA damage response independent of telomere length in mice. Proc Natl Acad Sci U S A 105:10173-8 (2008). WB; Mouse. PubMed: 18626023

Blickwedehl J et al. Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability. Proc Natl Acad Sci U S A 105:16165-70 (2008). PubMed: 18845680

Hudson CD et al. Brn-3a/POU4F1 interacts with and differentially affects p73-mediated transcription. Cell Death Differ 15:1266-78 (2008). WB. PubMed: 18421303

Blanco D et al. Molecular analysis of a multistep lung cancer model induced by chronic inflammation reveals epigenetic regulation of p16 and activation of the DNA damage response pathway. Neoplasia 9:840-52 (2007). IHC-P; Rat. PubMed: 17971904

Broom WJ et al. Inhibition of SOD1 expression by mitomycin C is a non-specific consequence of cellular toxicity. Neurosci Lett 393:184-8 (2006). PubMed: 16242241

Cheng W et al. DeltaNp63 plays an anti-apoptotic role in ventral bladder development. Development 133:4783-92 (2006). PubMed: 17079275

Xia F & Altieri DC Mitosis-independent survivin gene expression in vivo and regulation by p53. Cancer Res 66:3392-5 (2006). WB; Human. PubMed: 16585159

Hudson CD et al. Coexpression of Brn-3a POU protein with p53 in a population of neuronal progenitor cells is associated with differentiation and protection against apoptosis. J Neurosci Res 78:803-14 (2004). PubMed: 15532030



I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

Dear xxx,

Thank you for your support. After examination of the technical comments regarding this antibody, I've found any person experiencing problems with the same lot, so in such case I would like to try another lot. The details of the original order are:

-Purchase order number:
-Order reference number: xxxx
Please contact me if you need further information.

Best regards,

ANSWER:

That's no problem. I have now arranged for the antibody to be delivered to you. The new order number is xxxx(FOCR xxx).

I hope you find that this replacement works better than the previous vial. If you do have any further problems please do let me know. There may be a few things which could be optimised in the protocol used, for example, we usually characterise our antibodies using BSA blocking. Switching to this blocking agent can sometimes have dramatic effects on the results. An example of this can be seen on the datasheet of ab9385:

http://www.abcam.com/gapdh-antibody-hrp-loading-control-ab9385.html#GAPDH-Primary-antibodies-ab9385-3.jpg

The IHC experiment may also improve if the antigen retrieval method employed is optimised further. We typically recommend trying citrate buffer (pH6) and tris-EDTA (pH 9) at a few different incubation periods to determine which is most effective. More tips on this can be found from the following link:

http://docs.abcam.com/pdf/misc/abcam-protocols-book-2010.pdf

As I say, I would be very interested to know how you get on. Until then, I wish you all the best with your research.