your primary and secondary antibody dilution counts a lot in WB background issue. you may check /optimize that as well.

there are a few things that come to mind.

1. What membrane are you using-nitrocellulose or PVDF? In my opinion PVDF gives more background noise.

2. Your incubation time for your primary antibody is a little short. Generally, I incubate the membranes overnight at 4 degrees (unless it is a loading control such as b-actin which I incubate for 2 hours at room temperature)

3. What is the dilution of your secondary antibody? I would recommend 1:5000-1:10,000.

4. What are you blocking your membranes with? I would advise to always block the membranes in 5% milk TBST. If you are blocking in 5% BSA TBST, it is not as efficient and can lead to higher background. Even if you are using phospho antibodies, I would recommend blocking in milk and performing the incubations in BSA.

5. How long are your washes and what is the concentration of tween in your wash buffers? I perform 3 washes for 10 min each on a shaking rocker (60 rpm) and have a tween 20 concentration of 0.1%

You can try to play with the ECL reaction. We were forced to decrease the background noise by dilution of the substrate.

There are many ways to reduce background on blots

1. Dilute secondary Ab further and reduce incubation time: We usually use 20-30 minutes. Too long increases background

2. Use a more stringent buffer by increasing detergent concentration and/or blocking agent concentration. Use this for the wash cycles as well as Ab buffer.

3. Make sure blocking step is effective and your blocker does not cross react with secondary (or primary Ab, but more likely secondary). Block membrane then incubate with secondary Ab. Wash and develop to check. Change blocker if this is a problem. There are many ways to block a membrane: Milk protein, BSA are very common.

4. Make sure apparatus is very clean and transfer/SDS buffers are fresh. Any contamination will increase the chances of background

Possibly blocking issue. What are you blocking with? If you can, try milk for ECL (rather than BSA, which tends to give higher signal). Consider using some detergent in your washes/Ab incubations,, if you're not doing already doing it. And do not allow your membranes to dry as noise level goes up drastically in most cases.

Increasing blocking time may help. Moreover, you could use BSA instead of milk powder. Futhermore, the membrane type (nitrocellulose vs PVDF plays an important role for processing) etc.

Hi,

how is your background noise looking? Dots, smears, everything is overall dark?

Best,

Carsten

Chandra Somasundaram

I will try optimizing those, thank you!

Brandon A Kemp

1. I'm using NC membrane.

2. I will try increasing my incubation time

3. currently it's 1:2000, I will try altering the dilution ratio

4. I'm currently using 5% milk TBST

5. I perform 4 washes 15 minutes each on a shaker

Thank you for your suggestions!

Yuri F. Drygin

I will consider changing the ECL dilution, thank you!

Ian R Wilkinson

I'll make sure my blocking step is effective and my buffers fresh and new. Thank you!

Sébastien Soubeyrand

I'm using 5% milk in TBST, and make sure my membrane does not dry during the process. Thank you!

Andreas Eisenreich

I'll try increasing my blocking time, thank you!

Carsten Schmidt

Often what I observe is my band (which is relatively darker than the rest of the membrane), but the membrane itself it often while grey/or darker. And often there are dots as well. But I do not see much smearing or at all. I would ultimately like to optimize my protocol to be able to see nice, crisp bands with a less noisier (whiter) background (membrane). Thank you!

What are your antibody concentrations? For example, IMF is 1:50 to 1:500. WB will always run to 1:1000 or greater. No need to incubate at 4 degrees for your antibody incubations for the times you use.You also need to determine if BSA or a solution of dry milk is best for back ground. As Ian stated, too long an AB exposure the more chances are that background increases.