Edit: I have now managed to attached examples. These are both the same membrane, just different channels. I only see the dots when I use rabbit-800 secondary ab, and am now thinking it could be antibody aggregation. Would this be plausible? I will be spinning my aliquots down prior to using and will see if the dots disappear.
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I have done hundreds of Western blots with no issue, but I have recently started getting loads of dots on my membranes and can't seem to figure out why. They seem to concentrate more on the edges of the membrane, but will also occur in the middle bits of the membrane too. I use the Li-Cor system, and the dots are mainly just in the 800 channel.
Some background info:
I use either 5% milk, 1% milk or 5% BSA in TBST (0.01% tween-20) for primary antibody incubation and for blocking. I use 1:1000 dilution for primary antibody and incubate at 4 degrees overnight. For secondary incubation, I use the Odyssey blocking buffer (TBS) and the respective secondary antibody in 1:5000 dilution and incubate for 1 hour at room temp. I also filter all of my blocking solutions to minimize background dots.
Any suggestions would be welcome.
Looks like your blocking buffer is too old or it contains particles. try filtering the reagents before performing the same blot next time. if nothing helps try with an new membrane.
Thank you for your answer. I've used fresh blocking solution (filtered) and it's still causing problems. I even opened fresh batch of milk powder to be safe. I've ordered new membrane so hopefully that works better.
The things I could think of are
1) Bubbles during transfer, please make sure you pass a roller over the sandwich to get rid of those
2) Handling of the membrane, make sure it is not touched as fluorescence can pick that up as well.
Tuank you, they're not bubbles they're like black specs and I never touch with ungloved hands (mostly only touch with tweezers). I'm going to contact licor for suggestions too incase they can help.
Hi khushali - do you see those dots when you image your blot? if so, it is usually due to the secondary antibody; if it is not washed away properly the excess of peroxidase could still appear away from your bands and commonly it is in dot shape. I always wash my membranes in a large container, so even the edges of the membrane are washed. your secondary antibody is quite diluted, so likely you will not have a concentration Problem. If you actually see the dots on the membrane could be some sort of contamination from buffers?
Try centrifuge the antibodies 14.000rpm 2 min, especially secondary antibody. maybe it contains precipitate antibody. Prepare alwaysTBST with fresh Tween-20.
Noelia Dominguez Falcon thanks for your suggestions. I also wash in large containers and am very generous with the amount of wash buffer that I add. I also use fresh wash buffer so not likely to be contamination. Also this problem has just started recently, I have been doing westerns literally non stop for nearly 2 years now and all has been fine which is why I am so confused.
David Lopez so I usually keep a stock of 10% Tween-20 in deionised water in the fridge, and use that to make my TBST as I need it. I haven't had issues before but maybe I'll use fresh tween-20 and that may help. thank you
Hi Khushali,
Have you tried using another machine to develop your membrane, may be even on film (dark room). If that resolves the problem then there is probably nothing wrong with your technique but may be the machine. If it is the machine it could be an issue with the CCD camera seal, if that is the issue it might start condensation and crystal formation which could lead to the spots. Talk to the rep Li-Cor they should usually be able to trouble shoot over the phone with remote access.
Let me know if that helps!
Good luck :)
Priya Putta thank you for your suggestions. I am in talks with the Li-Cor reps ad they are trying to figure out if its a scanner issue. I think the real issue may be the brand of milk we are using, which was recently changed. Apparently using cheap milk in combination with rabbit secondary antibodies can lead to black dots. I will try the old brand again and see if they disappear.
Khushali Patel I see, if you believe its the milk buffer, I have a suggestion to switch to 5% BSA in TBST it works very well with my westerns and rabbit antibodies. BSA does take a while to dissolve so I recommend making it before hand and storing it in cold.
Good luck!
Priya Putta unfortunately today I saw the black dots on my membrane which was blocked with 5% BSA. I thought I had figured it out finally but now this happened so am thinking maybe its something to do with my secondary antibody as its the only common factor. Thank you for your help :)
Hello
kindly look at link here
https://advansta.com/wikis/high-background-in-western-blots-causes-and-solutions-2/
Good Luck
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