Hello,
I am actually doing a WB to identify the protein expression of PERK and IRE1, however I get really low intensity bands (I need to increase the contrast of the image a lot to see the bands and the image gets saturated) but I did a WB for GAPDH on the same membrane and it was working well as the image didn't have to be modified to see the bands. The transfer worked properly for the whole gel, the use of 100µg of total protein whether than 30µg didn't change the intensity of the bands, the antibodies are used at the dilution indicated in the datasheet, and a blocage has been made with milk. The membrane was exposed with the primary antibody during the night, and the 2ndary antibody linked with HRP was exposed for 1 hour. The ECL kit used will expire in 2018. What do you think the issue is and how could I improve the intensity of the bands?
Thanks in advance for anybody wishing to help!
is it possible to upload an image of your blot? Are you seeing bands for PERK and IRE1? If not, those antibodies may not be optimal for WB. Where did you purchase the antibodies?
Seeing the blot may help provide more troubleshooting tips.
Do you have a positive control? I have treated lymphoblastoid cells with tunicamycin at 2.5 micrograms/mL for 4 hours before and I could get good induction of ATF4. I think PERK and IRE1 will be adequately induced as well with the same drug treatment.
It is possible that your PERK and IRE1 antibodies are not working very well. If you expect them to work, maybe try a lower dilution. If the primary antibody solution has been sitting around in the fridge for a while, prepare a fresh dilution or expose for two days at 4 degrees Celsius. I find that those methods generally help.
I am agree with Liang Wei Wang
And you can do primary antibody exposure time at 72H at 4 degree celsius.
Best of Luck, Yann
I would like to suggest to make sure about your protein assay that you used to measure the protein in cell lysate . The good western result depend on many things ,such as accurate protein quantification in cell lysate, and the amount you should load in the gel and primary antibody concentration, and incubation time, and the blocking is very important step, you have to make sure if you prepare the blocking buffer correctly
Good luck and best wishes
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