Anti-INCENP antibody (ab36453)

Hi,

Recently we purchased ab36453 antibody. In the data sheet, it is written that there is 50% glycerol already added. However, the antibody freezes in the freezer. Is this normal? Normally we add 50% glycerol to avoid freezing if there’s no glycerol already in the antibody. This time we did not because it is already there. Should we add more glycerol? And why does the antibody freeze with 50% glycerol?

Kind regards,

ANSWER:

Thank you for your inquiry.

50% glycerol solution do in general not freeze through at -20C but will freeze at -80C.

If the freezer is a little colder than -20C the vial might freeze.

I would like to ask you to use the antibody in your experiments. In case the antibody does not work as stated on the datasheet, I would like to reassure you, that it is covered by our Abpromise.

I hope I this information is helpful. Please do not hesitate to contact em again with any further questions.

I am writing you as regards some of the antibodies of your catalogue that we bought recently: - ab36453 Rabbit polyclonal to INCENP (Order: 175095) - ab11054 Mouse monoclonal [2H12/4] to DMC1 (Order: 179315). We have tried these antibodies in mouse testicular cells but we have been unable to find any labelling in immunofluorescence. As the technical indications of these antibodies clearly stated they were applicable for immunofluorescence in mouse, we would be pleased if you could indicated us how to proceed in this case.

ANSWER:

I am pleased to inform you that I have received just this morning a detailed protocol for staining with ab11054. The antibody has been very satisfactory in detecting the Dmc1 protein on mouse meiotic chromosomes.

The meiotic chromosomes spreads were performed as described in the protocol that I have added to the online datasheet (accessible via the link below). Which means cells were fixed using a 4% paraformaldehyde, solution and permeabilized using SDS (0.03% in a 4% paraformaldehyde solution). The IF localization of Dmc1 was then conducted as described in the attached protocol. The 2H12/4 antibody was diluted 1/100 In 500µl of 1X ADB and incubated overnight at 4ºC. The secondary antibody used was a Goat anti-Mouse antibody labeled with Alexa-488 dye (A11017, Invitrogen), diluted 1/500. The slides were then mounted in a drop of Vectashield mounting medium with DAPI (H-1500, Vector Laboratories).

This procedure allowed localization of the Dmc1 protein as numerous foci in the Leptotene and Zygotene stages of meiotic prophase, and fewer foci in the early Pachytene stage of meiotic prophase.

I hope this information will be useful, please let me know if you still experience problems,

I am writing you as regards some of the antibodies of your catalogue that we bought recently: - ab36453 Rabbit polyclonal to INCENP (Order: 175095) - ab11054 Mouse monoclonal [2H12/4] to DMC1 (Order: 179315). We have tried these antibodies in mouse testicular cells but we have been unable to find any labelling in immunofluorescence. As the technical indications of these antibodies clearly stated they were applicable for immunofluorescence in mouse, we would be pleased if you could indicated us how to proceed in this case.

ANSWER:

I'm very sorry for the delay in finding out more information to help you and to hear you are experiencing problems with two of our antibodies.

I have been informed that ab36453 has been used in the following reference: Geddis AE & Kaushansky K. Megakaryocytes express functional Aurora-B kinase in endomitosis. Blood 104:1017-24 (2004). PubMed: 15130946 I enclose the staining protocol for this antibody:

ICC/IF: 1/1000. Fix cells in suspension by the addition of an equal volume of 4% paraformaldehyde prepared in phosphate-buffered saline (PBS, pH 7.4) for a final concentration of 2% paraformaldehyde. Fix cells at room temperature for 45 minutes and then centrifuge and wash twice with PBS. Resuspend cells in PBST (PBS containing 0.5% Triton-X100) with 3% bovine serum albumin (BSA) and incubate at room temperature for 15 to 30 minutes to provide blocking against nonspecific antibody reactivity.

Unfortunately I am still trying to find out the staining protocol with ab11054, I apologize for the delay. I would like to offer for me to look at your protocol as I may be able to make suggestions to improve the staining. I enclose the link below which will take you to a short questionnaire. If you please put it to my attention I will be able to continue my investigations and hope to help you resolve this problem:

http://www.abcam.com/index.html?section=ihc&pageconfig=technical&intAbID=11054&mode=questionaire

I look forward to hearing from you and hope the protocol for ab36453 will be useful,