I'm getting a ton of non-specific bands in my westerns.
My procedure is as follows, and I'm using 25 ug of protein per lane - the bare minimum I think I can get away with.
1. Ponceau S stain
2. Wash 3x 5 mins + 1x 15 mins in TBST
3. Block in 5% BSA in TBST
4. Wash 3x 5 mins + 1x 15 mins in TBST
5. Incubate in Primary 1 hr at 4 degrees (1:1000 in 1% BSA in TBST)
6. Wash 3x 5 mins + 1x 15 mins in TBST
7. Incubate in secondary 1 hr at 25 degrees (1:10,000 in 1% BSA in TBST)
8. Wash 3x 5 mins + 1x 15 mins in TBST
9. Detect.
The antibodies that I'm using are relatively new, and have produced clean blots in the past. The problem with the non-specific bands comes and goes, seemingly without rhyme or reason.
My primary antibodies target JNK, phospho JNK Thr183, PKC alpha, phospho PKC alpha Ser657, STAT3, and phospho STAT3Y705.
Lysates were prepared in either invitrogen Cell Extraction buffer supplemented with pierce protease inhibitor tablets and PMSF, or in CelLytic M with pierce protease inhibitor tablets and Sigma Phosphatase Inhibitor Cocktails 2/3 at 1% v/v. Samples were prepared with freshly made 5x sample buffer, and were heated at 95C for 5 mins immediately prior to loading. The problem occurs in a fashion seemingly independent of sample collection procedures.
I've attached pictures of a few problem blots. Your suggestions are greatly appreciated.
Dear Carl,
It is a very common problem and I guess everyone who does Immunoblotting faces it quite often. I think you need to standardize your conditions a bit. There are steps which when modified might help in circumventing this problem.
1. Like suggested sample preparation. I use boiling for 10 mins at 95ºC.
2. Your protein concentration is bare minimum, so nothing to do with that.
3. For the SDS gel, I would suggest you can resolve it a bit more so that you get only those non specific bands, which are very close to your protein's molecular weight. For example, if your desired protein is of 50 KDa, try to run off everything below 35 KDa. In that way you'll reduce the occurrences of lower molecular weight non specific bands. You can also alter the gel percentage for better resolution, so that, even if there are non specific bands they will be resolved away from your desired one and you can still be able to see the results.
4. Next step is transfer, but I don't think altering those conditions would be of much help.
5. Next, for blocking, I use 5% skim milk in TBST or 3% BSA in TBST. I generally keep for blocking for an hour. Then if I have used milk, before adding primary antibody, I give a brief wash just wash away the excess milk particles so that I doesn't interfere with your primary antibody's interaction.
6. Then add primary antibody (diluted in either TBST or BSA depending on blocking reagent used). You can dilute the antibody (1:2000 may be) and see if it helps. For incubation time, I usually incubate at 4 degrees overnight, but that depends on the type of antibody and its dilution.
7. Then I would suggest you to give 3 times washes each for 10 mins as already suggested by Miguel R. Guerreiro.
8. Then add secondary antibody whose dilution and incubation time can also be altered. I would suggest to use a higher dilution and lesser incubation time which would give the antibody just enough time to interact with its cognate protein but not enough for cross reaction.
9. Then before developing also you can give 3X washes for 10 mins each.
Hope it helps!
Dear Carl,
It is a very common problem and I guess everyone who does Immunoblotting faces it quite often. I think you need to standardize your conditions a bit. There are steps which when modified might help in circumventing this problem.
1. Like suggested sample preparation. I use boiling for 10 mins at 95ºC.
2. Your protein concentration is bare minimum, so nothing to do with that.
3. For the SDS gel, I would suggest you can resolve it a bit more so that you get only those non specific bands, which are very close to your protein's molecular weight. For example, if your desired protein is of 50 KDa, try to run off everything below 35 KDa. In that way you'll reduce the occurrences of lower molecular weight non specific bands. You can also alter the gel percentage for better resolution, so that, even if there are non specific bands they will be resolved away from your desired one and you can still be able to see the results.
4. Next step is transfer, but I don't think altering those conditions would be of much help.
5. Next, for blocking, I use 5% skim milk in TBST or 3% BSA in TBST. I generally keep for blocking for an hour. Then if I have used milk, before adding primary antibody, I give a brief wash just wash away the excess milk particles so that I doesn't interfere with your primary antibody's interaction.
6. Then add primary antibody (diluted in either TBST or BSA depending on blocking reagent used). You can dilute the antibody (1:2000 may be) and see if it helps. For incubation time, I usually incubate at 4 degrees overnight, but that depends on the type of antibody and its dilution.
7. Then I would suggest you to give 3 times washes each for 10 mins as already suggested by Miguel R. Guerreiro.
8. Then add secondary antibody whose dilution and incubation time can also be altered. I would suggest to use a higher dilution and lesser incubation time which would give the antibody just enough time to interact with its cognate protein but not enough for cross reaction.
9. Then before developing also you can give 3X washes for 10 mins each.
Hope it helps!
As an option, try to reduce primary antibody incubation time. Many commercially available antibodies are not very specific and might bind to different proteins.
Hope it helps.
Thank you all for your replies. I should clarify that the 30 minute wash step in the question is actually 3x 5 minute washes and 1x 15 minute wash, and that the blot is washed following incubation in the secondary. I've edited the question above to reflect this.
As for sample preparation, I'm presently running under denaturing conditions. The samples are heated in a block at 95C for 5 minutes.
There seems to be a consensus that I'll be better served by using milk in place of BSA when I'm blotting for the total protein, which I'll try ASAP. As for the phosphoproteins, using milk to block produces a black filter. This is unfortunate, because as you can tell in the image, the phosho-specific antibodies are the ones that are giving me the most non-specific background.
Could I block in 5% BSA in TBST and then do the primary (phospho-specific) in 1% milk without blacking out the filter?
Also, I'm quite a fan of Hamadi's suggestion of altering the primary incubation time - my current incubation time is 60 minutes. I used to incubate overnight, but I found a 1 hour incubation dramatically reduced the filter background. I'm going to give it a shot with a 20 minute incubation.
Also, I've considered changing my blocking procedure altogether - perhaps blocking in 5% BSA in TBS rather than TBST, or maybe trying a completely different blocking agent (again, I can't use milk). If you all have any suggestions I'd be thrilled to hear them.
I've basically given up hope with the STAT3/pSTAT3 antibodies after hearing from another researcher that good primaries for STAT don't exist. I was using STAT antibodies from Rockland Immunologicals, and I heard the ones from Abcam are terrible as well. If anyone knows of a good STAT3 antibody, I'd love to hear about it.
I found this guide guide to be quite helpful. See the attached file
I can suggest Stat3 antibody from Cell Signaling (cat. no. 9132; Rabbit mAb anti-Stat3.)
I agree with Fatma: the best WB antibodies are from Cell Signalling. Also, they provide you with a protocol on the antibody sheet.
Regarding your blocking step: milk works the best! For phospho proteins though, you have to wash the milk away before incubating the antibody. Incubate your antibody mix in 5%BSA in TBS-T.
I do have 1 question: What type of samples are you using for WB? Because if it is anything else than cell culture your background may be simply IgG/IgM.
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