In my experience these issues often come down to simple technique, and most the steps taken to keep things clean. Artifacts will likely spring from any number of sources, but here are a number of common ones (1) cleanliness of gel casting apparatus, (2) freshness and cleanliness of the membranes (PVDF or nitrocelluose), filter papers, sponge pads and buffers when transfering, (3) failing to cover the membranes during blocking, washing, and antibody incubations [dust falling into solutions and wash solutions during long incubations can be a huge issue in certain lab spaces], (4) contamination of the blocking agent [we don't use any tool to scoop the milk into the weighing boat, we pour it out carefully so that the remaining unused powder is kept clean and dry], (5) lumpy block from not mixing enough and leaving chunks of milk powder in the block, (6) dirty cassettes, plastic foil and so forth during ECL development [ECL loves dust for producing signal], (7) the choice of ECL kit and antibody concentrations - using what knowledge you might have about the likely protein expression to choose appropriate sensitivity of the probe reagents - i.e. an insensitive (weak) ECL will potentially leave you needing to use over long exposures which will bring out any background present.... I think from this list you'll note that in the main the cleaner, neater and more stringent you can be through out the process the better the results turn out. Good luck solving the issue, and happy blotting.

It could be due to the undissolved milk that has excessively blocked the membrane. Now, it is giving non-specific background. I had the same issue with my blots before and then I started to dissolve completely the blocking solution by keeping it on the magnetic stirrer for atleast 15-30 min. this will make sure that there are no undissolved particulate matter from the milk.

Also, you can try to filter the blocking solution and then use. Though, it is tedious but gives good results.

Now, I haven't experienced this problem in my blots.

Hope this will help.

Another problem can be bacterial contamination in the sponges (in case of tank blotting) or old blotting buffer.

Hi, another problem can be molds - filtration of all solutions (0.22 or 0.45 mikroM filters) will help also in this case.

Did you use beta mercaptoethanol for stripping, incase of harsh stripping this is a common problem is you dont was the membrane well........also check your exposure sensitivity, and as said above by others undissolved milk or other components may be a problem!!

I agree with Manuj.

You could try to use completely new solutions and check if you still got the problem. Then try the milk stirring, and if it doesn't work, try filtering it.

Best wishes

Maybe you could post a picture of the film?

I completely understand your problem since we were fighting with same issues in the past (black spots all over the membrane with no regularity). I agree that in some part its related to blocking solution aggregates BUT we tried I guess 20 different blocking solutions and no improvement was observed.

But finally we discovered that the problem was 2nd Ab-HRP - we've changed the vendor and the problem dissapeared completely and we have for the moment 2 years of spots-free lab life :-)

I hope that helps

P.S. Try to centrifuge/spin down the Ab before use.

Hi

According to my experience it can be due to:

1) quality of PVDF paper and film (expired may be)

2) time of and degree of incubation

3) High concentration of Ab in diluted liquid

In our case that was not a problem of 2nd Ab conc - we tried serial dillution method with no possitive effect.

All the best

MS

I had dots too once with a particular antibody against beta-catenin. For me it was a matter of switching from using TBS+Tween in all my washes and blocking and ab dilutions to PBS+Tween. Maybe something for you want to try in your Western Blot.

Did you wet the membrane before the transfer in 100% methanol for 15 seconds, or until the membrane

appearance changes uniformly from opaque to semitransparent? Equilibrate the membrane for at least 5 minutes in the transfer buffer. Perform the transfer and after that I briefly rinse the membrane with Milli-Q water and let it air dry.

You may dry the membrane and wet the blots in 100% methanol for 15 seconds. The blot will uniformly change from opaque to semitransparent. Proceed with blocking and immunodetection.

I've recently had a problem with random spots resulting from ECL, so I am paying close attention to this discussion. We recently switched to a new, more sensitive ECL substrate, so I attribute the problem to the increased sensitivity. I also suspect the milk used for blocking. We have bought new dried milk and I make a point to make it fresh each time (with plenty of stirring). What seemed to reduce the spots somewhat was storing the ECL reagents (in this case, Advansta WesternBright ECL) at room temp instead of at 4 degrees C. The reagent label specifies either refrigerated or room temp, so I figured room temp storage was less likely to produce any precipitation out of solution. I also feel that extensive washing with TBS-Tween after stripping is critical, as residual SDS or beta-mercaptoethanol could be causing the milk/blocking solution to curdle. Hope this is helpful...

Try changing the blocking agent from NFDM to BSA..i guess it would work fine in your case. Also, as suggested by others..ensure that your wash step is perfectly done.

In my experience these issues often come down to simple technique, and most the steps taken to keep things clean. Artifacts will likely spring from any number of sources, but here are a number of common ones (1) cleanliness of gel casting apparatus, (2) freshness and cleanliness of the membranes (PVDF or nitrocelluose), filter papers, sponge pads and buffers when transfering, (3) failing to cover the membranes during blocking, washing, and antibody incubations [dust falling into solutions and wash solutions during long incubations can be a huge issue in certain lab spaces], (4) contamination of the blocking agent [we don't use any tool to scoop the milk into the weighing boat, we pour it out carefully so that the remaining unused powder is kept clean and dry], (5) lumpy block from not mixing enough and leaving chunks of milk powder in the block, (6) dirty cassettes, plastic foil and so forth during ECL development [ECL loves dust for producing signal], (7) the choice of ECL kit and antibody concentrations - using what knowledge you might have about the likely protein expression to choose appropriate sensitivity of the probe reagents - i.e. an insensitive (weak) ECL will potentially leave you needing to use over long exposures which will bring out any background present.... I think from this list you'll note that in the main the cleaner, neater and more stringent you can be through out the process the better the results turn out. Good luck solving the issue, and happy blotting.

It sounds like a bacterial contamination ... or big aggregates in your solutions.

Use fresh solutions and filter them and move the membrane during all incubation steps continouslely with a shaker.

Did you wash the membrane with hot SDS solution, then let it equilibrate with your solution before applying blocking buffer again. A lot of times the membranes are not as effective the second time around due to aggregates that form on the membrane from the first probing. Also, as Andrew mentioned, make sure that the environment you work in is dust free and that your milk solutions are completely dissolved. The background on the membrane is higher since you have already used one complex of primary and secondary to probe the proteins, I would suggest washing the membrane with hot 2% SDS for 30 mins and try probing again.

Dirty sponges in transfer aparatus? Wash in hot water and try again.

I have had this problem in the past also. What worked for me was swapping from a milk block to a BSA block (the milk can clump and I think has impurities) and increasing the wash volume after stripping, to ensure all B-ME was gone (as per suggestion below). Good luck.

As others have commented, I've found that the black spots were usually caused by undissolved milk protein / precipitates that bind to the membrane and cause z-stacking that will bind antibodies non-specifically. You can avoid this by allowing the dried milk to dissolve for longer / thoroughly vortex the blocking solution, or filter the solution using a 1 um filter (best practice). Don't keep your washing solutions in large tubs at room temp for too long either - fungi can grow in the solutions and cause a blotchy effect on your membranes also.

I am using PBS+T and I have repeated stripping/ Reprobing using two different primary antibodies, yet I still get these random nonspecific spots. Moreover, I made sure that the NFM is completely dissolved before using it in blocking. Furthermore, I have made the best I can do to avoid dust particles during ECL. Despite all these steps I still get spots with variable numbers in different locations of the membrane. I will post an image of some of the results. N.B. the PVDF membrane was used previously and it gave good results. I do not know what is that I am doing incorrectly that resulted in these spots.

I have recently a problem with a lot of black dots which are randomly distributed on my membrane .I used NFDM 5% and commercial ECL from Thermo. I have also tried to spin down of 2Ab before used, but no progress was seen and I can say the blocking buffer was well dissolved. Does anyone have any advice.

Those spots do not appear if the PVDF membrane is stained with Ponceau's Stain. it only appear after ECL and on the X-ray film. Therefore, I tried to troubleshoot by making sure that NFDM5% is completely dissolved yet these spots still show up randomly every time. I tried to make the mambrane rest for a while face down to hopefully dilodge any possible particulate matter causing this artifact. This some times minimizes the spots but it is not enough to eliminate them completely. Therefore, I really appreciate any ideas to help me rid of this problem that has been ruining my results lately. Thank you everyone

Here in please attached an image of the X-ray film after ECL of the PVDF in WB. The Arrows indicate the black non specific spots in the lower part of the image and the non specific bands which do not coincide with any protein bands. The non specific spots in the lower part tend to scatter randomly on the film and they appear at different places when the experiment is repeated. Moreover, they are more obvious after stripping and reprobing the PVDF membrane. I have made sure that the 5 % NFDM is well dissolved before blocking the PVDF and I paid attention to avoid drying out of the PVDF in whole or in part during the process. Moreover, the membrane is relatively fresh that rules out any contamination or growth of microorganisms on the PVDF or in the buffer used in the experiment. Yet, I still see these horrible spots that ruins my results. I would like to ask you please for any suggestions as how I could get rid of these spots to achieve clean results from my experiments. Thank you.

I have the same problem with DIG southern blot many black spots and also the bands do not appear is all samples in spit of I have the same amount of DNA concentration

The non-specific bands look to be leakover from the adjacent lanes. This sore of thing happens with such sensitive techniques that we use. The spotting could be due to electrostatic shock. I haven't seen that in awhile, but that is what it looks like (electric discharge onto the film).