Hi! I got this western blot of the heat shock protein hsp70, and I need to interpret the result. The western is made on 2 individuals A and I and is shocked 5 minutes and 15 minutes, plus there is a control. But I am having a hard time interpreting the results. There should be more proteins after 15 minutes because the protein increase in number when stressed. How do I see that here?
Thankful for help!
To get results, you have to quantify against your control. Use ImageJ to see if there's an increased intensity in the bands and that's how you can say there's an increased expression. You also have to check if your antibody shows bands at different molecular weights as a non-specific binding or otherwise you have to check if they're breakdown products.
By just looking at this though, the increased expression at 15 in the second individual is clearly higher than the first 2 time points.
HSP70 forms antiparallel dimers which would come up higher than 100 kDa. It's possible you're seeing what you want to be seeing, but your western didn't properly denature/reduce your sample. Are you sure you didn't accidentally run a non-denaturing gel?
Jordan R. Yaron Hi! My teacher did the blot, so I believe he must have thought about that..?
@MuhammadAliHaidar Hi! So is it possible that antibody has attached to 100 and 25 kDa even though blocking was done? Or why are there bands outside of 70kDa?
I have a few points to make
1. Did you run to check the loading (e.g. actin) to quantify properly
2. Hsp70 during heat shock the protein concentrates strongly in nuclei while a small quantity remains cytoplasmic. Ref: Cell, Vol. 36. 655-662. March 1984.
Is your sample preparation homogenous? Does a nuclear vs cytoplasmic fractionation help?
3. It looks like two extra-thick bands around 120 in the 15min samples. Are samples properly denatured?
4. You can run a negative and positive control
5. You can use lower concentration of the antibody to reduce non-specific binding
Elina Qvist Yeah it is very much possible. So what would help is checking the catalog number of your antibody and checking what bands show when using it. I usually do that because a lot of the times, antibodies show unidentified bands that are irrelevant to your experiment.
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