Anti-CENPA antibody - ChIP Grade (ab33565)

CENPA antibody - ChIP Grade (ab33565)

Hello ­ I¹m trying to identify a CENP-A antibody for ChIP experiments in mammalian species. I¹m looking at ab33565, a rabbit polyclonal. In the description of the immunogen, it appears that residues 100-cTerm were used. In the customer feedback, there are immunofluorescence images of mouse and human cells that do not exhibit centromere staining. ( http://www.abcam.com/index.html?pageconfig=reviews&intAbID=33565)

There is a comment from Abcam on this: The immunogen used has no homology to the Human CENPA protein and therefore we would not expect this antibody to perform well under these experimental conditions.

What I¹m trying to ascertain is what abcam knows about the reactivity of this antibody and its utility under experimental conditions. The statement about the lack of homology is surprising ­ human and mouse CENP-A share significant homology in their C-terminal regions.

It is very important to be able to use reagents that are fit to purpose, as you know.

1. Can you clarify the situation with this antibody 2. Does abcam have a Œfitness to purpose¹ programme that allows an experimentalist to verify that the antibody Œworks¹ without financial risk?

Thank you,

ANSWER:

Thank you for contacting us.

The immunogen used to raise ab33565 is from 100 to the C-terminus of Mouse CENPA. The immunogen compatibility search between two species shows that only 5 immunogenic residues are shared between the two species, which is below our limit of 6 for cross reactivity. The immunogen sequence homology between two species is only 50% which is below than our recommendation of 90% for an antibody cross reactivity. That’s why we have responded to a customer in Abreview that this antibody would not be suitable for use with human samples.

We have an Abpromise for our customers which states if a product does not work in listed application and species than the product cost can be refunded. Full details about the Abpromise can be found by clicking the given link;

http://www.abcam.com/index.html?pageconfig=abpromise

More information about product use limitations can be found in terms and conditions of Abcam by clicking following link.

http://www.abcam.com/index.html?pageconfig=terms

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

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 262268

DESCRIPTION OF THE PROBLEM No signal or weak signal at 1:1000 dilution in 2% milk/PBS Signal for right size band (~17kDa) when used at 1:500 in PBS+BSA (no milk) but also background bands including H3 and H4

SAMPLE Mouse Liver Chromatin extracts

PRIMARY ANTIBODY CenPA from Abcam 1:500 in PBS+BSA or 1:1000 in 2% milk PBS 2hrs (RT) or overnight (4C)

DETECTION METHOD Li_COR at 700nm

POSITIVE AND NEGATIVE CONTROLS USED Magic Markers showed up fine (detectable by secondary antibody)

ANTIBODY STORAGE CONDITIONS Antibody sent on wet pack and left in Fedex truck over weekend. Delivered to us Monday at room temperature 3 days after shipped

SAMPLE PREPARATION Boiled in 1X LB + B-ME

AMOUNT OF PROTEIN LOADED 30 micrograms of chromatin per lane

ELECTROPHORESIS/GEL CONDITIONS 4-12% NUPAGE gradient gel, 150Volts

TRANSFER AND BLOCKING CONDITIONS overnight transfer at 100mAmps at 4C. Blocking in 10% milk PBS

SECONDARY ANTIBODY IRD_labeled anti-rabbit IgG, 1:10,000 o/n at 4C. 3 washes in PBS before scanning at 700nm in LICOR

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No

WHAT STEPS HAVE YOU ALTERED? Tried changing concentration- low concentration gives no signal, high concentration gives background

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you are experiencing difficulties with this product ab33565 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered.

I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. If the suggestions do not prove to be helpful, would you please be so kind to confirm the following items in order to help me better understand the cause of the problem. If you can also provide an image that would certainly help a lot.

Most customers who use milk as a blocking agent tend to have problems detecting bands. At Abcam, almost all of our products have been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. Furthermore, cross reaction between the antibodies with the diluent could also cause the lack of signals. We would normally use 1% BSA to dilute the primary and secondary antibodies. You can also use PBST only.

Therefore, I would suggest blocking with 5% BSA for 1 hour at room temperature (RT), and using 1% BSA to dilute your antibodies. Please try using 1:1000 for the primary antibody as a starting dilution and incubate overnight at 4C. For the secondary antibody, you can try a dilution at 1:300-5,000 but incubate at 1-2 hr at RT. At Abcam, we recommend using around 20-40ug per well of protein sample. If the above recommendation still show multiple bands, you may want to lower you protein concentration to 20ug/well or lower.

Can you confirm you have reduced and denatured the sample at 95oC for 10 minutes in buffer containing SDS and mercaptoethanol? This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody.

Can you confirm the washing buffer concentration? How much Tween was in the PBST solution? In general, a 0.05% PBST would be sufficient as a washing buffer. Also, was the washing done 15min x 3 times? This is because over washing will eventually cause you to lose signals. While, insufficient washes will cause high background.

I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with your shipping address/purchasing agent information. Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.