Please see attached 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 during the process.
Yet, I still see these horrible spots that ruin my results. I would like to ask you please for any suggestions as how I could avoid these spots to achieve clean results from my experiments. Thank you.
Hi, Mohamed
ECL is so sensitive, have you tried with chloronaphthol substrate? Try to do washing times more? did you use Tween 20 in the washing buffer? I work with semi dry (BioRad) for blotting, may be the transferring procedure is not correct,
Good luck
That may be some unwanted substance such as sticky stacking gel and undissolved NFDM etc. on the membrane. Sometimes the glove or dirty forcep will introduce dark spot on the blot. Here is a link for your reference.
http://www.abcam.com/index.html?pageconfig=resource&rid=11352#5
Good luck.
If you use any blotting paper or paper towels during your process you might want to change the supplier. Also, you have to keep those things sealed from dust.
Another matter is the building of the blotting sandwich. Be sure to avoid ANY tiny bubble. Depending on the kind of film exposure you use: use another type of foil or plastic bag for your membrane. Avoid multiple layers of foil.
How do you do the incubation? In a chamber or in a plastic bag? Exchange that procedure as well.
Do you cast your own gel or using pre-cast gel? Do you use any petroleum jelly when assemble the gel glass plate?
Hi, I cast my own gel and I do not use petroleum jelly for assembly. I wash the glass plates with tap water and soap, then I rinse them in distilled water and finally I wipe them with 70% Ethanol. I have noticed that these spots become more notorious when I strip the PVDF and reprobe it with another antibody. I wish if I could know what makes them become that way to avoid it and be able to reuse the PVDF of the same experiment over and over as this more convenient and saves time and money for me.
In my opinion, those non-specific bands that found most probably because of the nature of antibody. Are you using polyclonal antibody? What is your protein of interest? Then what kind of container you use to hold the membrane for washing and antibody incubation?
I use polyclonal antibody and I use a plastic container for washing as well as incubation of the PVDF. I noticed that it is more prominent after I strip the PVDF and re-probe it; therefore, I am thinking that it could be due to residual charged SDS that nonspecifically bound to the antibody. Yet, I usually wash the PVDF for 2.5 hours after stripping to eliminate any trance of the stripping buffer I do not know what else I should do to avoid appearance of these spots.
Polyclonal antibody may just bind non-specifically on other protein even the protein in your marker lane. for me that's not a big problem.
SDS less likely is the problem. The signals become more obvious after re-probe may solely because of the affinity of antibody.
Try to solve the spot by :
1) Using new or clean transfer solution when blotting.
2) make sure your plastic container is clean. Try with 70% alcohol, detergent, distilled water and last rinse with TBS-T before use. Autoclave it can make things better (If your plastic container can be autoclaved)
Try to use a different blocking agent such as BSA or Caesin
Never allow the membrane to dry especially between transfer and blocking, and while changing buffers,
If you are using trays to wash, add buffer before putting the membrane
Dear Mohamed Alalem,
At least some of the spots in your film could be due to static electricity in the film or in the plastic that you use to cover the blot while exposing it to the film. When the static electricity discharges, it produces light and thus you see a spot in the film. I cannot explain, however, why you should have more static electricity after stripping and reprobing the blot... Anyway, I had the static electricity problem once with electron microscopy films and it looked similar to your spots. In my case it was due to drying the film before loading it to the microscope casettes. We got rid of the spots when we started drying the films AFTER loading them to the casettes. In your case, you could try storing the films in normal room atmosphere, i. e., not in a special dry place (unless you are doing to already).
Somehow the blot with weird bands in the bottom gives me a impression of being mishandled or mistakenly touched bare hand? Hope you follow the basic principles of WB. There are interesting comments above, hope they help you explain your problem. I use fluorescent based detection and the bands are very clean. My suggestion would be
to change the blocking buffer to BSA and wash it more frequently with wash buffer (PBST/TBST). In addition, you can prepare your antibodies in 2-4% BSA in TBST/PBST. For me washing several times more frequently worked well when i did chemiluminescence.... good luck!
The way or the protocol you perform the wb is not ideal: 1st to use the gel loading tips to load the protein from bottom to up to avoid the donuts shape of the bands, 2nd make sure to load equal volume each lane in 1x sds buffer; 3rd transfer/blotting at 4-8 oC and make sure you have the ice cooler inside the transfer box; 4th use 2x or 5x less of secondary antibody you are using now.
You also should change your concentration of the secondary antibody. I think its to much HRP on the blot.
I would dissolve your NFDM - the black spots are something I used to get a lot, and then someone told me to gently pre-warm the NFDM @37oC for 30m prior to use - this removed the black spots. Also you could consider using a non-milk based blocking reagent, this would increase the useage amount of your antibodies too.
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