Unidentified problem with low concentrated samples

[ggerard]

Hi there,

I'm just trying to stablish a protocol for Western Blot for my proteins. I am working at the nanogram scale. The problem is that I've been able to reach the 1 and 0.5 ng limit of detection two times, but for any misterious reason I'm having a lot of troubles with reproducibility.

I have no idea of what it can be, but I've tested a lot of details. I am using polyclonal antibody as the 1st, anti-rabbit-biotin as the second, HRP-strpt for the enzyme, and the supersignal femto substrate from pierce.

I've modified the concentration of all things, tested different washes, etc. However, I cannot find the problem. The last experiment, today, I've done 4 membranes. The 1st and the 3rd were identical, but the third employed re-used 1st pAb. Well, only the 3rd membrane has shown the 10 ng band (the more concentrated one, not seeing the other 5, 2 and 1 ng bands). I mean, the fresh antibody works, I saw the 1 and 0.5 ng with it, there is not any kind of logic here!

The transfer works well (I see the coloured proteins in the membrane and a clean SDS-PAGE gel, semy-dry transfer).

So the problem is, I reached a low limit of detection (1 ng) using a protocol. When I repeat the protocol, I've been able to reach that limit only one another time. Trying to find the problem, I've changed a lot of things, it appears that despite any changes, I have an aleatory problem that I cannot identify.

I suspect of how I add the femto substrate, but I've changed it for different "practices" without any difference. I also have a lot of background problems, that I have not been able to reduce by incremented washed.

So, any idea?? Thanks!

[ggerard]

Further details.

Main solutions:
1st antibody, 5 g/l
2ng antibody, 1 g/l
HRP-strep, 1 g/l

Dilution used:
1st antibody, the maximum I can, 1/2.000 (suggested 1/50-1/500)
2nd antibody, I've tested 1/2.000 and 1/20.000
HRP-strep, I've tested 1/400 and 1/8.000

And none of these combinations have resulted in a stable protocol. I've also tested milk blocking at 5 and 3 %, the 1st antibody incubation is overnight 4 ºC, although 1h at RT has been also tested.

Hope some of you have any idea, because I just do not know what can be the problem ...

[butters]

well for 1st antibody do u freeze and thaw?

incubation @ 4'C i never did before so i really can't comment there unless u r looking for cold antigen-antibody reaction. i would try 3h, 8h @ RT but u must make sure the membrane is wet and agitate all the time.

For concentration i would usually do dotblot as i can control better amount protein load per dot.

finally what is the size of ur strips and how much volume of antibody did u use?

[ggerard]

Nops, is the same aliquote, stored at 4 ºC and it is yonger than 1 month. The incubation at 4 ºC overnight is to obtain the maximum signal. However, today I've done two membranes with 2h at RT. I've seen clearly the 10 and 20 ng band, but the 5 ng is very subtle. The two membranes were identical but one with re-used 1st pAb. This one (very strange, but this is the third time I see a similar effect) has provided slightly better bands.

I'm using very exact boxes in terms of size, half-membrane (4*7 cm then) with 4 ml of solution. Visually, it is enough, I think I could actually add only 3 ml.

The protocol that I have followed today is different, and I have the intention of from this, trying to improve the sensitivity (the same I did in the past, now with an obsessive control of the changes). 1st pAb 2h RT, 2nd pAb-biotin 30m RT, HRP-strp 30m RT (in PBS, last wash also in PBS). Dilutions, 1/2000, 1/25000, 1/25000.

Now I'm thinking about increasing the 1st pAb concentration to 1/800, but I have doubts in increasing any of the pAb-biotin or HRP-str concentration and/or incubation time.