Anti-ATF6 antibody (ab37149)

Thank you for your answer. Unfortunatly, at the moment I think that the antibody does not recognize the 50kd for some reason, as it doesnt work in my positive control. The main interest of people in ATF6 as an ER Stress marker involves the regulation of its cleavege and monitoring the amount of the cleaved protein. That is why i think that it is very important for you to show that your antibody can recognize the cleaved part. If you do have new information regarding this issue, i would be very happy to be informed.

ANSWER:

Thank you for your reply. Unfortunately we do not know of a suitable positive control for the 50kDa band and have yet to test the antibody for the cleavage fragment. I hope that the recommendations I sent to you regarding protocol changes will help you detect the 50kDa band (use of a RIPA buffer, boil samples in loading buffer at 95-100C for 5 minutes, changing the blocking step, incubating the primary antibody overnight at 4C more concentrated, use Ecl+ detection kit and check the secondary antibody).

Good luck with your experiments,

BATCH NUMBER - ORDER NUMBER -

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE glial cells

PRIMARY ANTIBODY 1:2000

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED -

ANTIBODY STORAGE CONDITIONS 4

SAMPLE PREPARATION I attach the western results, and here are more details: I use mice glial cells (raised in primary culture), and I induce the ER stress by incubating cells with thapsigargin (0.5 micromolar for 20 hours or 1 micromolar for 3 hours. After washing the cells with PBS I lyse them with this buffer: 20mM Tris-Hcl (PH=8), 10mM NaCl, 1mM EDTA, 0.1% Triton, 0.5% SDS, protease inhibitor cocktail: 1:500 and Phosphatase inhibitor cocktails (1:100). After vortexing for 15 sec, I centrifuge (in 4 deg) for 30 min in 14,000 and took the soup. After boiling in 70 deg for 7 min, I loaded 50 microgram of protein (+Sample Buffer) in an SDS-PAGE gel – 10%.

AMOUNT OF PROTEIN LOADED 50 microgram

ELECTROPHORESIS/GEL CONDITIONS 10%

TRANSFER AND BLOCKING CONDITIONS 5% milk, for 1 hour

SECONDARY ANTIBODY goat anti-rabbit-HRP

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No

WHAT STEPS HAVE YOU ALTERED? -

ADDITIONAL NOTES -

ANSWER:

Thank you for your enquiry. I am sorry to hear you are having a problem with ab37149. Unfortunately the attachment you have mentioned in your form did not reach us, could you please send it to my attention so I can better understand your problem?

I would like to already suggest the following modifications to your protocol:

-run a positive control, as it may be that currently your lysate does not contain high levels of the transcription factor. The antibody was originally tested on MDA MB 361 cell lysate so this is a recommended positive control.

-As the protein is located in the ER and in the nucleus, I suggest you switch lysis buffer to a RIPA buffer, as this is stronger and will extract the protein very effectively, increasing its levels in your lysate.

-We recommend to boil samples in loading buffer at 95-100C for 5 minutes. It may be that currently the heating to 70C is not sufficient to effectively denature the protein, hence it is not recognized well by the antibody.

-Try blocking with 5% BSA as some antibodies bind to the milk and hence do not bind to the membrane.

-Incubate the primary antibody overnight at 4C. It would be worth trying more concentrated antibody, for example dilutions of 1:500, 1:1000.

-We recommend to use Ecl+ or more sensitive kits such as the super signal Pierce kits, as we find that ECl is not very sensitive. Please let me know if this helps and do not hesitate to contact us for further advice.

-Check the secondary antibody works with other primary antibodies, as it may be responsible for the low signal if its HRP activity is diminished.

I hope these recommendations will already help you, please do not hesitate to contact me if I can be of further assistance.