I don't have further tips to share on top of what has been already said. But I remember two things that killed my blots and took me some time to figure it out. 

One was the presence of sodium azide in the antibody mix. I just added to preserve a dilution of a precious homemade antibody. Blots were not working again and after some time a colleague told me that the NaN3 inhibits HRP reaction. When I used an aliquot from the original undiluted stock everything went back to normal.

The second was when I tried to blot actin and an actin binding protein in the same blot. Somehow, and don't ask me why, the antibodies seemed to neutralize each other. They were a mouse monoclonal and a rabbit polyclonal. When used separated both work perfectly....

Hopefully these anecdotic comments shed some light.

Claudia is right, moreover you have to check you HRP solution, sometimes there is a problem with the H2O2, or you have some technical issue with the exposure time, try to increase the sensitivity and the time of the exposure but as Claudia said first you have to examine you antibodys, actin or tubulin will be a good start

good luck 

The antibodies work very well, the HRP, the ECL developing puffer and the exposure time. We share these with other in my lab. The problem is I'm running the same samples that I had perfect blot two months ago and they are stored in the same place as my lab mate samples. 

Hi John,

How do you store your samples? Lysate alone or already in some Laemmli-like buffer? Are you trying to detect phospho-proteins?

As mentioned above by Claudia and Konstantine the first thing you have to be sure about is antibody functionality and ECL/HRP conditions. But if your labmates are able to get good blots using the same antibodies/ECL stocks than you (meaning the very same stocks) and buffers are at a correct pH then the usual suspect is that the sample deteriorated with the time by degradation or losing features like phosphorylations. This shouldn't happen when your samples were stored already in Laemmli-like buffer or any other mailing enzymatic activity. 

Also you can check the blocking agent you're using. Some blockers can mask antibody epitope specially when antibodies get old and their title is reduced.

I stored my samples in Laemmli-sample buffer and to confirm that they are good I made new samples but the result is nothing. Also, we use the same blocking buffer. I'm trying to detect total Akt and b-actin which are abundant in my cells.

Make all the chemical freshly and then use again....if not coming then may be antibody degraded due to thawing activity....

In my experience, the same blocking buffer does not behave perfectly the same, since there are many littile things that can change, as evaporation, oxidation, and many other factors......Usually when this problem occurs, your antibody get stuck on anything

So I am oriented to think that the membrane you used is being exposed to much on oxygen (if you take of the protective leayer your blotting membrane will be unpredictable after half a day or at maximum few days) and/or other environmental factors

But more often the blocking buffer is not being assemble/diluted correctly or prepared too much in advance in respect of the blocking of the membrane, a very common problem

Dear John,

Please check with below steps.

·         Use a higher concentration of antibody; incubate longer (e.g. overnight) at 4°C

·         Use an enrichment step to maximize the signal (e.g. prepare nuclear lysates for a            nuclear protein, etc.)

·         Do not over-wash the membrane.

·         Use fresh antibody as the effective concentration is lowered upon each re-use.

·         Use fresh substrate

Hope this info helps you.

As the previous answers attest to, there are 1,001 ways to fail with Western blotting. So controls and testing of each step is key to finding your problem on any given day. It sounds from your answers so far that the issue is in the detection, not electrophoresis or transfer of samples. One possibility to consider is that your first result was wrong somehow and your reagents are not up to the task of detecting the proteins in your sample, but somehow you saw bands earlier, maybe non-specific ones, that now are gone. If you cannot detect actin then this should be the focus of your attempt to resolve the issue. If other people in other labs are getting good data with the same sets of reagents, then I suggest that you ask them to blot with your actin Ab and systematically compare things with them until you find the reagent that is defective. Good luck. 

Aside from all the answers already posted, you may just want to make sure your connections are correct, red with red and black with black. It could be that your proteins are running off the gel and into the buffer well beforehand and thus you will never see any bands.

I too thought of this but the proteins were found on the membrane after transfer, thus I believe the problem must lie downstream of the transfer. Everybody connects the electrodes wrong once in their careers. Hopefully only once...

Yes, I stained the membrane with Ponceau stain and my proteins are there.

You might also want to check the lot# on the antibody, especially if it's a polyclonal. There can be some lot-to-lot variability on the efficiency of antibody binding (as well as the ug/ul antibody concentration) and you may need to increase the amount of antibody you are using and incubation time. Also, as noted by some others, freeze-thaw cycles of the antibody and sample can make a big difference. And it would be a good idea to make up EVERYTHING fresh (WB sample, Ab dilutions, blocking buffer-if it's made in house, check that they haven't changed the brand of BSA or milk, and ECL reagents). I always keep my own aliquots of Abs, etc, since you don't always know if someone else in the lab may have flaked out and left an antibody or sample sitting on their bench overnight or accidentally cross-contaminated Abs or other reagents.

I don't have further tips to share on top of what has been already said. But I remember two things that killed my blots and took me some time to figure it out. 

One was the presence of sodium azide in the antibody mix. I just added to preserve a dilution of a precious homemade antibody. Blots were not working again and after some time a colleague told me that the NaN3 inhibits HRP reaction. When I used an aliquot from the original undiluted stock everything went back to normal.

The second was when I tried to blot actin and an actin binding protein in the same blot. Somehow, and don't ask me why, the antibodies seemed to neutralize each other. They were a mouse monoclonal and a rabbit polyclonal. When used separated both work perfectly....

Hopefully these anecdotic comments shed some light.

If you can see the bands   with Ponceau stain , you  have problems with the antibodies  (expecially with the secondary labeled Ab) or with the ECL reagents.  Check their quality and,  about the ECLreagents ,   be sure that they were not  contaminated by  using the same tip!

Hi,

     It may be your secondary AB is not working properly... 

I haven't read all the other suggestions, but if you are sure about you protein samples and the transfer as seen by ponceau staining then there may be a problem with primary or secondary antibody or the ECL reagent that you are using. 

i) You may probe your samples with some other antibody- loading controls such as GAPDH or actin- to verify their integrity.

ii) Try using a separate lot of primary and secondary antibody.

iii) Be sure that the ECL reagent is working. Borrow ECL from someone whose western blots have developed properly.

Good Luck

Hi Sama,

I see already many good suggestions. My first idea was: did you possibly overload your gel with respect to your specific protein band? This came to my mind because you see it upon ponceau staining, which is usually far less sensitive as compared to ECL. Having too much HRP-bound antibody associated with the specific protein may use up the substrate to quickly in the surrounding area and create "negative" bands/spots. Just an idea - hope you solve your problem soon.

If your primary antibody and the secondary antibody are the same as before, and they are not denatured, may be the substrates that you are using to detect are not right. Is it alkaline phosphatase or HRP based or fluorescence/enhanced chemiluminescence based detection system? Please check the substrates by in vitro reaction.

Do you reuse your antibodies solution?  after thrice or four uses they ussually dont give signal due to denaturing by repeated freeze thaw.

Thank you all for your inputs. I found that I have contamination with some minerals in my final step and that's what's killing my HRP.

Did you check the concentration of your antibody? It may varies among the lots.

If you usualy use an arbitrary dilution like 1/1000 or 1/500, be aware that in your solution, your antibody could be more or less diluted depending on the stock solution concentration. Cell Signaling for exemple provides lots with very different concentration for the same antibody.

Some times the blocking agent can mask antibody molecules.

I have the same problem, I'm able to detect B-actin, but can't detect p-AKt or total AKT in my samples. have try different secondaries

If the materials are all good and not degraded/expired, the machine to read the signal set well as no other operators altered the wavelength filters or any other setting which could make you blind to your reading.

I would check the blocking conditions which are primary if you block too much no antibody will bind.

I would then check the signal emission as for exampe your using a substrate to activate an enzyme on the antibody or any other way. It could be that the substrate is not working.

If the substrate is ok, I would check the washing stringency.

Strong advice for future!! Use IR emitting antibodies, they lasts half a year or more on dried membranes and they emit when excited by IR. Very few problems in my experience with that antibodies! Very recommeneded !!!

what do you mean when you have contamination with some minerals in final step where it kills your HRP? Where is the source of mineral and how do you able to figure that out? Thanks! Sama John