Hi Paula,

You may try using 5% BSA in place of Milk as blocking and Ab dilution step.

Milk may be creating some aggregates during the block step and causing those nonspecific patches to form on your blot.

Good luck,

Hey Paula Peressini ,

From my experience when using LiCOR fluorescent antibodies for western blots, I was getting high background or dots like you have posted when I used milk. I tried BSA and had a similar effect. For me what resulted in the cleanest blots while using these secondaries was utilizing LiCORs blocking agent (its expensive but worth trying to troubleshoot). So the main thing is your time vs money. If your time is more valuable than purchase the PBS-Licor blocking reagent buffer. If money is the worry than try to optimize with BSA instead of milk. Just from my own work with it, the LICOR blocking buffer worked even better than BSA. See my example of quality below

Hey Shawn Krueger, Thanks for your answer! Just to be sure, do you mean doing the blocking + 1ary Ab incubation also in LiCor Buffer? I incubate the 2ary in LiCor buffer always.

PS: I only get this nasty stuff with the green anti-Rabbit. The red doesn't seem to do that

Hi Md. Areeful Haque and Sudeep Kumar thank you both for your answers. We actually don't use BSA because in the past has given high background compared to milk.

Paula Peressini ,

So for me I used the the PBS based blocking buffer for: Blocking, primary incubation, and secondary incubation ( https://www.licor.com/bio/reagents/intercept-pbs-blocking-buffer ) and then for normal washes I used fresh PBS.

Non-fat milk powder is a complex mixture of proteins and hence a better blocking agent than the homogeneous BSA. However, because milk proteins are phosphorylated, they can't be used for anti-phosphoprotein antibodies. Try fish skin (teleost) gelatin instead, that tends to give very clean blots.

The spots look like your blotto may have contained particles, for example of undissolved milk powder. Give it a good spin (say, 10 min at max speed) and then use the supernatant.

We have had similar problems previously, we put it down to insufficient solubilisation of milk protein prior to use: When mixed well on roller mixer problem went. Milk protein changes upon long term storage and becomes more difficult to solubilise so use a new batch and re-test. In your blot is could also be particulate matter in the gel (not clean plates/solutions) that attract Ab or even small pieces of gel sticking to PVDF membrane post transfer?

Would blame your gel rather than the Ab. Probably tiny pieces of gels that stick to your membrane. Make sure your gel is well polymerized and relatively fresh (if you make it yourself). Your gel shouldn't stick to membrane after transfer. In any event very unlikely due to be Ab or blocking related (would result in more uniformly high background). Have you tried secondary Ab only? Or even no Ab (straight after blocking)? Could be due to fluorescence/acrylamide based artifacts. Also, I may be mistaken but Li-Cor suggests to block without Tween; could give it a try but would only help with uniform background issues. And agree with above, Li-Cor reagent works well but milk is typically quite good as well (and BSA is typically not quite as good); Ab dependent to a large extent.

Hi, Paula Peressini and all others.

I did not work with LiCOR but one thing I may add to all other comments is take a look and clean the recipient where you washing and blocking your membrane. Sometimes, If you reuse it several times, some molecules and rests of old solutions (antibodies, fluorescent/luminescent reagents, milk, proteins, etc) you used may stick around and go to your next membrane reaction. It's quite simple to solve and inexpensive ... Of course, If you use some one-use recipient to do this stuff, just keep other people's option in mind.

Last thing, About milk or all other blocking reagents you have been talking about, you may pass it through a syringe membrane in order to get rid of what is not solubilized. Ok?

Hope it helps.