Unidentified problem with low concentrated samples - Variable limit of detection of my western (Dec/19/2008 )

Hi there,

I'm just trying to stablish a protocol for Western Blot for my proteins (~ 20 kDa). 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 2 membranes, cut after the blocking step into two parts.
1) fresh pAb (1/2000) + pAb-biotin (1/2000) + HRP-strep (1/1000)
2) fresh pAb (1/2000) + pAb-biotin (1/2000) + HRP-strep (1/8000)
3) re-used pAb (1/2000) + pAb-biotin (1/2000) + HRP-strep (1/4000)
4) re-used pAb (1/2000) + pAb-biotin (1/8000) + HRP-strep (1/4000)

To note, I saw the 1 ng and 0.5 ng using fresh pAb. I've began to use re-used pAb recently after 2 months of experiments and wasted reagents.

Suggested dilutions, 1/50-1/500 for the 1st pAb, 1/10.000-1/100.000 for the pAb-biotin and HRP-strep.

Samples loaded, 10, 5, 2 and 1 ng lanes.

Then, only the membrane 3 have provided a nice vision of the 10 ng band. How can I explain this? All the membranes have been processed in paralel without any difference.

I have no doubts of the transfer since I see the coloured proteins in the membrane. In all membranes where I have charged 50 ng I've seen them. With 10 ng in some cases (like today) I've not seen any band. Some times I can see the 5 ng bands. Just 2 times I've reach the 1 ng and 0.5 ng limit. I have applied the same protocol but it is not reproducible.

I suspect of how I add the femto substrate. I've tested directly, from a pipette, incubation, it seems not to affect. Neither the number of washes. I have high background, but actually I do not relate it with the fact of viewing the bands or not. When I see the bands the membrane is dirty, but the marks are clear.

So, what can be the problem?? I have no idea of what can it be. The next gel I will dilute the substrate to do an incubation, and I'll try to use less biotin and streptavidin reagents (I already did test it). Is the pAb concentration so important? 1/500 is suggested, I started with 1/40.000 and I have been decreasing it.

The complete protocol:

SDS-GEL 15% acrylamide, 250V in 40min (doing at 150V makes no difference), 55 mA during 30 m (all the marker transferred to membrane, an Immobilon P-sq), buffer TRIS with methanol, no SDS, membrane activated with MeOH and transfer buffer, gel incubated with transfer buffer for 10 min, membrane blocked with 5% milk (tested also 3 %) 1h RT in PBS (18 ºC), pAb incubated in 1% milk overnight 4 ºC, washed 3 min x 3 or 5+5+10+10 min (no difference) with 0.5 % milk, pAb-biotin (I've tested 2,5 and 20 µl in 40 ml) in 1 % milk, 1h RT, the same washes, HRP-strep (tested 5, 10, 20 and 100 µl in 40 ml), washes with PBS (3 min x 3 or 5+10+10+10+10 min, no difference), add 1 ml of supersignal femto substrate mixture from the eppendorf directly to the membrane, cover with plastic, read.


Absolutely no idea of what can I do more. The membranes are already cut and 3 months old, when I saw 0.5 ng, 1 month ago. Sincerely, I do two identical membranes at the same time, one is ok, the other not. The last success, I could see 5 ng last week, 2 identical membranes, the first with fresh pAb, the second with re-used pAb, I saw 10 ng with the fresh pAb, and 10 and 5 ng with the re-used pAb. How can it be explained??

Please, any help will be appreciated. Thanks!

Gerard,

Your observations could be explained by assuming that 5 ng is very close to the limit of detection, and the variability is explained by measurement (e.g. pipetting) errors. All pipettors have a measurement accuracy and reproducibility range.

There are also minor inter (and intra)-run variabilities such as circulating buffer in the transfer, slight heat differences during the run and transfer, all of which can affect the end result.

Reusing antibodies when you are approaching the detection limits of a system is probably not a good idea as even slight differences in the concentration and/or degradation could be the difference between detecting and not detecting.

First of all, thanks for the responses.

HomeBrew, pippettes are calibrated, and it wouldn't explain these differences. I mean, If I want to put 10 ng of a protein taking 5 µl, I can actually take 4 µl due to an error in the pippete, so instead of 10 ng I would put 8 ng. The range of these mistakes does not explain what I amb observing, this is in the same day with two identical membranes, only in one I observe the 10 ng band (clearly), in the other nothing.

Chimaera, you're right with the milk and biotins. However, in all my experiments the milk has always been present (always the same source of milk), so whatever is causing this variability should not be the milk. However, today I've changed the procedure to adopt this suggestion (thanks). Now HRP-strep is incubated in PBS without any milk. The results have been optimal in terms of background noise (I've reduced as well the amount of 2nd pAb and STR-HRP), however, I have seen (clearly) the 10 and 20 ng, but the 5 ng band is very subtle.

Bob1, these minor variabilities that you mention could be the reason, but it is difficult to demonstrate. However, temperature is strictly controlled in the lab. I am always worried about the rpm in the mixer, too fast? too slow? but I do not believe in a high impact of these details (I do not discard it, however). I am using re-used antibody just for replicates. For example, the experiment of today, for the 3rd consecutive time, the bands with the re-used pAb were more defined than the when using fresh pAb. Not a lot more, but it is very strange. How the antibody can give better results after having been used before?

I have changed the protocol today, in order to minimise the background noise. I've seen 5 ng with difficulties, 10 ng without problems. 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.

I am thinking now what I could change to increase the sensibility from this point (trying to see again the 0.5 ng). I'm thinking about decreasing the 2ng pAb dilution to 1/10000 and increasing the incubation time from 30m to 1h. However, in theory these quantities should be in excess, so the only that I will get is more background noise. But perhaps 30m is too little time and I am reducing the sensibility. Any opinions on this?

And a last question. How do you add the substrate (femto supersignal) to the membrane? 1 ml directly over the membrane? You do an incubation with 5 ml? The plastic cover is necessary for something? How many time do you give to the reaction before you read?

Thanks in advance!

Note: I upload the image of the membrane of today, and the membrane where I could read down to 0.5 ng. The protocol followed there was different, yes, but I have repeated the same protocol many times not repeating the same results. I do not consider an error of the concentration of the solutions, since I used the same in the posterior experiments.

Perhaps your protein is not completely solubilized? You are assuming a homogeneous solution where every aliquot by volume contains the same amount of protein -- this is only true if your protein is completely in solution and does not aggregate...

Finally, after dozens of tests, the background problem appears to be only caused by the 1st antibody (strong evidences). I guess that all the other variables have a little impact on the final results. Indeed, a higher concentration of the antibody does provide a lot of background, while less quantity reach the same detection level with less noise. So I've reduced the pAb concentration.

However, I cannot find why in two times I could see 1 ng, while now I see just 10 ng, sometimes clearly, sometimes not clearly.

And furthermore, I know (I am totally sure) that the background noise is also produced by how I add the femto supersignal (pierce) substrate to the membrane. Now I am drying the membrane previosly (placing over a paper) and adding the substrate with a pipette. However, I can see some waves that I suspect (heavily suspect) are caused by the pipette-substrate deposition.

Today I am running one of the lasts (or probably the last) tests, with higher tween 20 and milk in the 1st pAb incubation. If this does not work, I will change of antibody ...

Could it be that the primary is actually degrading slowly, if you are storing it in the fridge this is a strong possibility. We store most antibodies in aliquots at -20 or -80 deg C, even if the product sheet says to store at 4 deg C.

Sometimes you will see an improved result after the first time you use an antibody because the initial use has removed tiny amounts of inhibitors and secondary/nonspecific bands that are causing the high background.