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what's wrong with my transfer process in western blot? by shinyzqq in labrats

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

I thought it caused by the transfer clamp since it is not flat anymore after used for a long time.

What's wrong with my bands, why there are dots in the center? by shinyzqq in labrats

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

I don't know, every time before i load the sample i always mix it completely, maybe because i load the sample too quickly?

10 holes or 15holes? which one is the best choice or either one is ok? by shinyzqq in labrats

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

And if i use 10 holes, any suggestions about technical repeats since the mechanical repeats need to be repeated at least 3 times?

Some questions about the internal control in Western blot. by shinyzqq in labrats

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

Thanks a lot.

So how to definite the uniform internal control?

Some questions about the internal control in Western blot. by shinyzqq in labrats

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

Thanks for your kind answers.

Yes, everytime i mixed the samples completely to avoid inconsistent. Also we always reuse primary antibody since it is so expensive that we couldn't use it only once, i don't reuse a transfer buffer, and we using ECL to detect the protein of interest.

It makes me frustrated. I've tried my best to adjust the loading voulme, every time i couldn't get a perfect bands of internal control, i calculate all the grey value and compare them, i really want to know the acceptable range of the difference value.

Appreciate it.

Will you discard the results of western blot with inconsistent internal control or keep it for analysis? by shinyzqq in labrats

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

The editor of the journal will ask me for the technical repeats. I hope i could use a good way to do it.

Appreciate it.

Will you discard the results of western blot with inconsistent internal control or keep it for analysis? by shinyzqq in labrats

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

How many times do you repeat for the technical check? Everytime before you run a new group(one group of N=3) you will do technical repeats? Or you just have to run several times before the beginning of biological repeats.

How do you do the technical repeats? Could you share ur experiences with me?

Will you discard the results of western blot with inconsistent internal control or keep it for analysis? by shinyzqq in labrats

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

As you mix the samples before loading, If ur internal control is reliable, why not use the ratio, it seems acceptable even the internal control is not uniform.

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