Hello everyone,
I am getting a strong positive band from my ladder lanes. However, it doesn't even match up in size with any of the stained standards! It is showing up at maybe 60 kDa, between the pre-stained 75 kDa and 50 kDa standards. Picture attached.
I am using Bio-Rad's dual color standards (1610374), and the membrane that the band is showing up in was stained for human TLR4 with Bioss's antibody (bs-1021R). In the image, the second membrane below was probed for actin (Abcam ab1801).
Any ideas what could be causing this, or what steps to take to determine what it is?
Hi Kelly,
Have you talked to Bio-Rad people about this? Do you have the possibility of testing a different batch? Before to change anything you want to make sure that it doesn't come from your ladder sample.
Hi Kelly,
First, It doesn't seem to come from sample driping to the ladder lane because there is no match between them. Nevertheless I'd recomend to keep one empty lane between the ladder and the sample so you can account for that.
Second, I can't avoid noticeing the multiple bands you are getting on your samples, that could mean you are using a not very specific primary or secondary antibody that may be recognizing as well some protein in the ladder (that you can't see stained because of its small concentration)
If after asking BioRad they don't seem to know the why, it'd be good to use more protein in the sample lanes and less primary antibody in order to avoid unspecific recognition.
Thanks for the advice! Yes, I do have some nonspecific staining that I'm trying to work out. I'm waiting to hear back from Bio-Rad as well.
Reply from Bio-Rad:
"You assumption is correct, one of the standard bands contain a sequence to your polyclonal TLR4 Ab. The standards have very low level contaminating bands that do not show up with regular staining techniques, but, under the right blotting conditions will be visible. This is what you see. Unfortunately, you cannot really change this. You didn't see it at 1:5000 (your band either) which strongly suggests non-specific binding."
Very interesting -- I wouldn't think there'd be enough contaminating protein in the ladder mix for any signal to show up like this, but I suppose I was wrong. Bio-Rad suggested cutting off the ladder lanes and skipping primary Ab incubation for those lanes. But I think first I will try increasing protein loaded (from 10 ug to possibly 20 ug?) and decreasing primary Ab, as Edward suggested.
Thanks again for the replies, and have a great week!
Hi Kelly,
If I was you I would not worry so much about this band that shows up in the standard since you do not have it in your sample. It is something coming from the standard that is recognized by your antibody, nothing to worry much about. Better spend time improving the quality of you western blot as you may still have some non specific signal. As to make a figure, you do not necessarily have to show the lane with the ladder, often we simply report the MW manually on the figure.
Dear Kelly:
I am really interested in how you fix these problems in the end.
Cause one of my associates is facing the same problem (also got a band at 70-75 kDa in M.W. marker lane ) and suffered multiple bands.
And they are using polyclonal antibody as well.
Would you please help to advise on this case?
Your help would be appreciated. I’ll look forward to your reply
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