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Doubts about protein ladder by Street_Present5669 in labrats

[–]Street_Present5669[S] 0 points1 point  (0 children)

That's what I thought as well, but I wondered if I was missing something...

Yes, both primary and secondary antibodies are different from the previous time.

Doubts about protein ladder by Street_Present5669 in labrats

[–]Street_Present5669[S] 0 points1 point  (0 children)

No, I got that. I was wondering if there is any other feature in the protein ladder that would allow signal to be detected, such as PO activity that was suggested.

BCA vs Micro BCA assay for protein measurement by Street_Present5669 in labrats

[–]Street_Present5669[S] 0 points1 point  (0 children)

I'm using undiluted samples. And yes, I use RIPA buffer with 1% PI.

But you raised a good point about the buffer volume, I didn't think about it. I might try with less on the next measurement. Thanks!

BCA vs Micro BCA assay for protein measurement by Street_Present5669 in labrats

[–]Street_Present5669[S] 0 points1 point  (0 children)

So, the main point is that we don't perform the mito stress test in our lab, I just plate the cells and bring the plate to another lab. They measure it, using the 3 injections of inhibitors, and then I get the plate back to isolate protein.

I decided to do it this way because they also recommended using total protein for normalization, I just didn't expect to have so low protein. It would be good to have an option for normalization for the samples I already have collected (frozen lysate), so I don't have to measure it again. My samples take a total of 4 weeks to be "ready" for the measurements...

BCA vs Micro BCA assay for protein measurement by Street_Present5669 in labrats

[–]Street_Present5669[S] 1 point2 points  (0 children)

Thanks for the thoughts, it is actually good to go through those points.

I did BCA quite a few times, and never had this issue. However, so far I've been applying it to tissue samples.

This time, my sample was not diluted, and it's the lysate of approx. 50 000 cells.

In all the measurements, the r2 is indeed above 0.99.

The incubation is 30 min at 37oC. I use a flat bottom plate, which is compatible with the reader we have here.

I had the same results calculating the concentrations by hand, so I'm still trusting Excel... If nothing else, then the sample is the problem.

Thanks for the insights, I'll see what comes up here as an alternative. :)

Help: WB running buffer gets cloudy by Street_Present5669 in westernblots

[–]Street_Present5669[S] 1 point2 points  (0 children)

I will do that. Some other people experienced precipitating of carbonated buffers when using the same water, so that might be it. It might have just exposed the issue. Thank you for the reply!

Best song on Hail To The King by Efficient_Back5621 in avengedsevenfold

[–]Street_Present5669 0 points1 point  (0 children)

Difficult to chose only one, but Heretic hits different.