Anti-Cleaved PARP antibody (ab4830)

Dear sirs   I would be interested in buying the 2 antibodies listed below and to use them on bovine cells. Since I see that the anti caspase 3 one has not been reviewed yet in this species, would you be interested in getting it tested in bovine?   Anti-Caspase 3 antibody - Cleaved at Asp175 (ab52294) Anti-Cleaved PARP antibody (ab4830)

ANSWER:

Thank you for contacting Abcam with this inquiry.

Unfortunately the homology between the immunogen of ab52294 and bovine Caspase-3 falls below the threshold for qualification for our Abreview testing discount.

Please let me know if you have any questions or there are other way in which I might help.

Dear Sir/Madam

The customer would like to PARP antibody that result same with the publication (page 1387, attach file) which have PARP protein and cleave form. So, please kindly recommend abcam antibody.

ANSWER:

Thank you for contacting us.

We have several antibodies which can be used to detect PARP in Western blotting. From the study I believe you wish to detect both the cleaved (C-terminal fragment) and the full length PARP form in mouse samples? This can be done using ab75757 (Chicken polyclonal) or ab37722 (Rabbit polyclonal). Although ab37722 has not been tested with mouse samples due but to the similarity in immunogen sequence (100%) it is predicted to react.

If however you are interested in human samples this can be achieved with ab32071 (Rabbit monoclonal [E78]) which was raised against a synthetic peptide of the C-teminal residues of human PARP. Please note this antibody does not react with mouse samples.

We also have several antibodies that specifically recognise the cleavage product of PARP such as ab4830 (reacts with human C-terminal PARP) and ab32064 (reacts with mouse and human PARP, N-terminal).

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 167107

DESCRIPTION OF THE PROBLEM no band

SAMPLE Cell extract

PRIMARY ANTIBODY ABCAM/Rabbit/1:1000 and 1:500 dilutions in 5% milk/TBST(Also tried without blocking with milk at all---using TBST alone.

overnight incubation

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED ABCAM Beta actin purchased last year used as negative control.

ANTIBODY STORAGE CONDITIONS -20 degrees

SAMPLE PREPARATION RIPA buffer/PMSF

AMOUNT OF PROTEIN LOADED 30 micrograms

ELECTROPHORESIS/GEL CONDITIONS 10% gel (acrylamide gel)

TRANSFER AND BLOCKING CONDITIONS Transfer buffer: Tris base 48mM--5.8g, Glycine 39mM---2.9g, SDS 0.37g, 20% methanol---200ml to make 1L. Blocking agent: 5% milk. Blocking time: I hour

SECONDARY ANTIBODY Santa Cruz/Rabbit/5% milk/TBST/1:1000

Incubation for 3 hours at room temperature

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5

HAVE YOU RUN A "NO PRIMARY" CONTROL? No

ANSWER:

I'm sorry to hear you are having a problem with ab4830.

I did have a question about the samples you used. What kind of cells did you test? Were these treated to induce apoptosis? This antibody will only detect cleaved PARP, so if the cells are not apoptotic, the protein will not be detected (please see the second Western blot image, where the uninduced cells show no band, and the induced show a very nice band).

The rest of the protocol looks fine to me. It may just be that the sample you are using is not supposed to have cleaved PARP.

Please let me know if this helps and do not hesitate to contact us for further advice.