Anti-PON1 antibody [17A12] (ab24261)

What is the concentration of HEPES in the buffer?

ANSWER:

Thank you for your phone call yesterday, and I apologize that we did not already have this information.

I have heard back from the lab, and the buffer of ab24261 contains 10 mM HEPES.

I hope this information is helpful, but please let me know if you have any further questions.

I'd like to detect human PON1 protein expressed in the liver. But PON2 and PON3 are also expressed in the liver, so I need to use a PON1 specific antibody that don't cross-react to PON2 or PON3. Do you habe any information on the cross-reactibity of your 6 anti-PON1 antibodies with PON2 and PON3? Which product do you recommend for my purpose?　Thank you.

ANSWER:

Sorry for the long wait.

I have another update about one of the products you previously enquired.

ab26930: We have not tested this product for cross reactivity with PON2 and PON3.

ab24261: There is no reply from the originator and I would assume that they too have not tested the product for cross reactivity with PON2 and PON3.

I am sorry I can't be of much help in this occasion but I hope that the information for ab92466 was sufficient for you to use the product in your research.

If there is anything else that I can help you with, please do not hesitate to contact me.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE tissue extract

PRIMARY ANTIBODY abcam/mouse monoclonal/TBS/1:500 and 1:1000/1 Hour / overnight / 3 washes with TBST for 15 minutes .

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Positive control (Heela cells, plasma)

SAMPLE PREPARATION 50 mM phosphate buffer saline pH 7.4/ heating for 3 minutes at 100C

AMOUNT OF PROTEIN LOADED 60 ug

ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE/12% reducing gel

TRANSFER AND BLOCKING CONDITIONS transfer buffer(tris, boric acid, EDTA)/3hrs. blocking agent (5% Non-fat milk/5% BSA)

SECONDARY ANTIBODY [another company] / horse / TBS / 1:2000/ 1 hour / 3 washes with TBST for 15 minutes.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? transfer time (1 hr. and 3HR.)/anti body incubation time / anti body dilution / chnging blocking agent (BSA and non- fat milk)

ADDITIONAL NOTES we did not get any band in positive control (Heela cell , plasma) as well

ANSWER:

I'm sorry to hear you are experiencing poor results with ab24261 in western blotting.

Your protocol seems good in general and I have only a few suggestions which I help with resolve the problem:

-As you are using a good positive control of HeLa cell lysate the problem does not seem to be low expression levels in this sample, however it would be worth trying a different lysis buffer as currently the buffer you use does not seem to be suitable for optimal extraction of the protein. I would therefore recommend using 20 mM Tris-HCl, pH 7.5, 1 mM EGTA and adding protease inhibitors to the buffer. Following lysis and centrifugation I would recommend to add reducing loading buffer to the supernatant and boiling for 5 min at 100C. -I am not familiar with the use of boric acid and EDTA in transfer buffers. We typically use a Tris transfer buffer made with 20% methanol and I would recommend to try this type of buffer.

-it is good that you tried different blocking agents. Can I please make sure that you have tried blocking 1 hour only and incubating the antibody overnight in TBST only as sometimes the antibody binds the blocking agent rather than the membrane, if incubated too long together.

-it would be worth checking that your transfer is working adequately by doing a Ponceau staining.

I hope the suggestions above will help you. Please do not hesitate to contact me if those changes do not give you a better signal and I would be happy to offer you a replacement vial or refund if the antibody was purchased in the last 120 days.

I already purchased PON1 antibody. I would like to know it is rasied against what peptide sequence.

ANSWER:

Thank you for your enquiry. This product has been raised against the full length recombinant form of the target. I hope that this information helps.

It says that the antibody cross reacts with Mouse PON1. What is the epitope that the antibody recognizes, and how similar is the detection, ie: will 5?g of hPON1 give the same response with chemiluminescence as mPON1?

ANSWER:

Thank you for your enquiry. The antibody ab2461 has been tested in human samples of HeLa cell lysate and HepG2 cell lysate and in mouse samples on mouse liver tissue lysate on different gels therefore we cannot compare the amount of PON1 protein detected in those samples. In our hands the antibody works very well for both species,

I hope this information will help you, please do not hesitate to contact me again if I can be of further assistance,