For high-background antibodies 10% milk or 5% BSA ON should work at least in some cases. Also the combination of milk/BSA is a well blocker in case of problematic antibodies. But all of these innovations are requiring time-consuming set-up. In my eyes there is no simple solution for problematic antibodies, every single behaves different...

By the looks of it Rockland blocking buffer is probably just BSA with some preservative. You can just make your own 5% BSA. Works well for me. I was always told to avoid milk with the licor as it supposedly increases background.

5% BSA works well as a blocking buffer. For some antibodies, 10% skimmed milk is required for blocking. You have to standardize the use of blocking buffer whether to use bsa or milk based on your antibody.

5% milk worked for 95% of our western on the Odyssey. For the rest we either used BSA or rarely the Odyssey blocking buffer. When testing new antibodies we always started with milk.

Thanks for your suggestions,

It seems to be, there is no real good "homemade" alternative to the commercial "infrared adjusted" blocking buffers. Our lab tried them all from BSA to gelatine, all of those who work great for ECL. However, when we overexpress a protein, we also get reasonable results with the ECL type blocking reagents but endogenous .. bad, too high background. Obviously only Licor and Rockland are competing in this field?

Thanks a lot again

Michael

I have attached the image showing four different blocking conditions: 5% Milk in TBS, 5% BSA in TBS, Odyssey Blocking Buffer/TBS (1:1) and Odyssey Blocking Buffer /TBS (1:2) on nitrocellulose membranes probed with mouse anti-actin and goat anti-mouse800CW. They were all really nice with no background, however, the actin bands were weaker in Milk and when tested with the red channel (700nm), Milk gave rise to autofluorescence.

Hope this may help.

Heidi

Hello Heidi,

thanks a lot for sharing your testing.

I have a two questions:

1) Did you use ordinary nitrocellulose membrane? We are using a lower autofluorecence PVDF-membrane.

2) Have you experienced the same nice results for low abundant proteins?

Thanks again

Michael

Hi Michael

I am using the standard 0.2 µm Nitrocellulose membrane (Cat# 170-4159) from Biorad. After the transfer I incubate the blot in TBS for 5 min before adding the blocking solution. For the past 6 months I have used the Odyssey Blocking Buffer diluted 1:3 in TBS and get really nice results for both low and high abundance proteins, including phospho proteins.

I have tested different concentrations of Tween-20 during incubation with the primary antibody and see no difference between 0.05%, 0.1%, 0.2% and 0.4%. Now, I routinely use 0.1% Tween-20 during both primary and secondary incubations. I also have 0.01% SDS present during the secondary incubation. After the last wash with TBS+0.1% Tween-20, I wash the blot in TBS before scanning the image. Also, my TBS solutions are prepared from Trizma Base (pH adjusted to 7.6 with conc. HCl) and not as a mixture of Tris-HCl and Tris-Base. This also appears to give better results.

All the best,

Heidi

We use a very economical fishgel-based blocking buffer at .5% in TBS that works extremely well with IR, and does not leave the same background as milk and BSA... You do have to use the fluorescence-rated PVDF membranes however....