Reducing is a must.

Considering the amount of your starting material and assuming a reasonably efficient IP, by loading 25 to 50µl of the first eluate you will be able to see what you want.

Do not forget to neutralize the acidity of your eluates (you have likely done it since you are working with a kit). At first, I would try with 25µl ; if your IP worked you will see it.

You could also load 25 µl of your first and second eluates just to compare and to get a sense of how much is in in the second. You could also boil a fraction of your beads in Laemmli loading buffer in order to assess how much is still on your beads.

Thank you, Cirille!

I will try that (loading 25 ul of eluates). I think to run it in parallel to the initial cell lysate. I guess 25ul will be a lot. So, I will do dilutions, e.g 100%=25ul, then 75, 50, 25, 5, 1, 0.1%. Does it make sense?

If you want to load dilutions, I would go for 50% and 25%. I am afraid going lower will not bring anything. In theory, as little as 100pg of protein is detectable by WB, even 10pg for highly sensitive detection systems. This is how it should be in an ideal world...but we are not in an ideal word.

Hello,

Thank you very much Cyrille for you help.

An update. Hope it will be useful for somebody.

Yes, I neutralized the protein eluates with the solution from the kit. But.. it turned out it did not work. How do I know? I added 6x laemmli buffer (5 ul) to 25 ul of the sample and the dye in the buffer turned yellow.

To correct pH 1 ul of 10 M NaOH was added to the sample, which turned the sample colour back to blue.

The samples were loaded to the SDS-PAGE and.. it looks like there are no proteins in my eluates :((

Q could it be that my proteins now are under basic conditions and proteins do not separate?

Q or did the proteins degraded because of low pH?.. because if something else? (No protease inhibitors in eluates)

By the way, the original cell lysate (25 ul+ 5 ul 6x loading dye) and the lysate dilutions (1/2 - 1/32) we’re loaded on the gel (Lysate: 5*10^6 NALM-16 cells lysed in 500 ul of the lysis buffer from the kit). Lysate was visible on the stained gel to almost 1/32 of its dilution.

i did not boil used beads as they dried out.

1µl of NaOH in 25µl makes ~384 mM final, it is too much, when you add your leammli you cannot reach the pH 6.8 which is required. Once neutralized, the pH should be around 7.

But you cannot escape some troubleshooting. You will have to figure whether the IP has worked: analyze the flow through (supernatant) before and after IP by WB. If the signal of your protein of interest is as strong after than before IP then your IP hasn't worked. If the signal after IP is strongly diminished compared to before IP then the IP has worked. If so, you will have to analyze whether the elution has worked: best would be to analyze a small fraction of your beads before and after elution by WB. Same principle as described above etc...

it is a pain in the a.. but it is the only way to understand. everything can be done in one go.