Hi, From my experience with a weakly expressed protein, things started to improve seriously when i went for PVDF membranes instead of nitrocellulose and most of all tried different blocking and incubating buffers: PBS vs TBS, Tween vs Triton and milk types (even powder milk from 2 different brands made a difference). And finally the incubation temperature. I ended up with nice bands on a very weak background where I had dirty stained WB before with for example strong ECLs. (things have likely improved since, that was 10 yrs ago).

I'd recommend to make 2 rounds of setup, the buffers 1st to see which gives the less background. I used to test 6 conditions with a 15-well gel loaded with nice positive and negative controls and cutting membrane slices having each a half of ladder.

I'd incubate the primary antibody O/N at 4°C anyway and in a second round of tests try if 2° at 4°C too (O/workday for 6-hrs) improves, in my case it did a lot. The bands of interest were much clearer and sharper all the time with this whereas with 1H RT they could sometimes be so blurry you wouldn't see them clearly.

And also consider circadian variation in expression level, the amplitude is huge in the liver and some cell lines can be synchronized, a complete medium change can be enough (Article The 2008 Pittendrigh/Aschoff Lecture: Peripheral Phase Coord...

Good luck!

Dear Kakali,

In my experience, decreasing washing steps just leads to increased background.

What method are you using to detect your antibody? There are methods that can increase your sensitivity, such as the ABC method where you use a biotinylated secondary antibody followed by the ABC reagent that is complexed with an enzyme (I prefer HRP). The enzyme is then reacted with a colorometric reagent (like DAB).

If you are using a chemiluminescent detection method, you may just have to (carefully!) increase your exposure time to get a stronger signal. Are there any cell types that express higher levels of this protein that you can use to optimize your detection method?

I see that Cell Signaling has an antibody guarantee. I suggest contacting their tech support to see if they have any suggestions for your cell type.

Good Luck!

Jill

Dear Kakali Ghoshal ,

Are you using TBS-Tween 20? From my experience, reducing Tween concentration gives darker bands. Thus, I think you may try Tween concentration of 0.05% if you are using 0.1%. And also incubating and washing for too long might give less dark bands. Washing for 10 minutes with three changes worked fine.

This antibody is normally quite faint I usually use incremental detection with bands appearing at 4-5 minutes mark. What is your detection system/process? If it's chemoluminescence what ECL do you use? You should not use skim milk with Foxo (our lab eventually switched from milk to BSA for everything), however BSA 5% is quite excessive and expensive 2-2.5% is more than enough with 1% for antibody dilution.

Hi, From my experience with a weakly expressed protein, things started to improve seriously when i went for PVDF membranes instead of nitrocellulose and most of all tried different blocking and incubating buffers: PBS vs TBS, Tween vs Triton and milk types (even powder milk from 2 different brands made a difference). And finally the incubation temperature. I ended up with nice bands on a very weak background where I had dirty stained WB before with for example strong ECLs. (things have likely improved since, that was 10 yrs ago).

I'd recommend to make 2 rounds of setup, the buffers 1st to see which gives the less background. I used to test 6 conditions with a 15-well gel loaded with nice positive and negative controls and cutting membrane slices having each a half of ladder.

I'd incubate the primary antibody O/N at 4°C anyway and in a second round of tests try if 2° at 4°C too (O/workday for 6-hrs) improves, in my case it did a lot. The bands of interest were much clearer and sharper all the time with this whereas with 1H RT they could sometimes be so blurry you wouldn't see them clearly.

And also consider circadian variation in expression level, the amplitude is huge in the liver and some cell lines can be synchronized, a complete medium change can be enough (Article The 2008 Pittendrigh/Aschoff Lecture: Peripheral Phase Coord...

Good luck!

I agree, PVDF membranes may lead to better results. Additionally, you could increase concentration of primary and/or secondary antibodies, increase incubation time or perform over night incubation at 4°C, and/or use alternative blocking substrates (e.g. 3% BSA in TBST instead of 5% milk powder for 1 to 2 hours at RT of over night at 4°C). Moreover, the aforementioned comments seems also potentially helpful.

In any case your protocoll should be revised and adjusted in this context.

I also agree that PVDF is much better than nitrocellulose. All of the above suggestions are great ways to troubleshoot the problem. Interestingly, I also found the same problem with Tween that Muzaffar Muminov noted. I figured this problem out when, in desperation, I ran two blots side by side (actually one PVDF blot cut in half with duplicate bands on each half). One blot used Tween in the buffer, the other did not. The blot with the Tween buffer had absolutely NO positive bands detected by my primary antibody! The other blot was fine, with strong bands.

Also of interest is Daniel Pouly's suggestion of circadian rhythm. I work on the retina, and certain proteins have also shown changes in expression due to circadian rhythms, so I sacrifice my animals at the same time of day at all time points. I did not think of this in terms of cell lines.

Good Luck with all the troubleshooting tips-

Jill

Jill Miotke

Thanks for the suggestions.

I used Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate from Perkin Elmer and developed in Syngene. I also used odyssey system of Licor with their secondary antibodies. But in both cases the band is still faint. Odyssey system is little better sometimes than the ECL one. The exposure time for ECL is 3 minutes. I have tried from 15 seconds upto 5 minutes. And 3 minutes used to give me better band.

I generally incubate the membrane with primary in 4 degree overnight in 5% BSA in TBS. The antibody gives very strong non specific bands around 50. I tried to incubate with various concentration of Tween 20 (0.05%, 0.1%) but that did not give any better binding. So I avoid Tween 20 during antibody incubation since its specific binding is very weak. I contacted the tech team but they are not sure about liver and hepatocytes. They have different positive control.

I used to give 4 washes (5 minutes each in TBST with 0.1% Tween 20). After secondary incubation I give three washes with TBST with 0.1% Tween 20, 5 minutes each. Then the last wash in TBS without Tween for 5 minutes.

I might try PVDF membrane. I am using 0.2 micron nitrocellulose membrane.

If your method is correct or you didn't find better way in this research, an In Silico study to design and optimize antibody-receptor interactions and then synthesis of this more specified antibody is suggested.

From my experience PVDF > Nitrocllulose , primary antibody in BSA > skim milk, and for 24 + hours at 4 degrees. we also use PBS for our washes rather than TBS, and never seem to have any problems.

Wash times/number of washes generally only impacts the background signal, not the intensity of the detected band itself.

Increasing the secondary antibody concentration could be something worth looking into if you havent already tried? Or depending on your detection method, ECL prime can also be a good resource as it detects very faint bands.

Also, depending on your detection method (we use chemiluminscent) altering your exposure time may help detection.

Many variables to check...some suggested above. Check them one by one on a positive control and only change one variable in each experiment.

1. Compare 5% FCS to skim milk.

2. Agree regarding 24 hrs at 4C. but you can try different temps. . and times.

3. Compare TBS vs PBS.

4. Titrate both the primary and secondary antibodies..one at a time.

5. Vary exposure times.

We usually wash 5X but that is another possible variable.

Once the assay is optimized, you just have to go with it and always use positive and negative controls in each experiment.

It all in the antibody, try to replace secondary or direct labsl your antibody, there are fast and easy kits to label up to 10ug of antibody. All the washes, blocking, buffer can reduce backgrund .