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Endogenously Biotinylated Proteins


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#1 biologist2B

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Posted 12 April 2009 - 02:37 PM

I was wondering if I could use either milk or BSA to block my nitrocellulose membrane? I seem to have high background and non-specific binding.

What I did was blocked my membrane with 5% BSA in TBS for 4 hrs at RT. Then I blotted with streptavidin-HRP in TBS (no blocking buffer) for 2 hr at RT. Then I washed 4X, 15min, TBST (1mL of Tween per 1L of TBS). I added 1mL of SuperSignal chemiluminescent for and then imaged my membrane. There does not seem to be any changes among the lanes which indicates that the streptavidin is binding non-specifically.

I was under the impression that milk has endogenously biotinylated proteins and I cannot use that to block. I would appreciate any advice!

#2 klinmed

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Posted 13 April 2009 - 01:31 AM

I was wondering if I could use either milk or BSA to block my nitrocellulose membrane? I seem to have high background and non-specific binding.

What I did was blocked my membrane with 5% BSA in TBS for 4 hrs at RT. Then I blotted with streptavidin-HRP in TBS (no blocking buffer) for 2 hr at RT. Then I washed 4X, 15min, TBST (1mL of Tween per 1L of TBS). I added 1mL of SuperSignal chemiluminescent for and then imaged my membrane. There does not seem to be any changes among the lanes which indicates that the streptavidin is binding non-specifically.

I was under the impression that milk has endogenously biotinylated proteins and I cannot use that to block. I would appreciate any advice!


In my experience blocking a membrane with milk does not increase the background in biotin/streptavidin systems. This is in marked contrast to its effects on detection using some lectins or phosphorylation-specific antibodies.

Eukaryotes contain few endogenously biotinylated proteins (usually carboxylases) and these are usually mitochondrial and absent in milk. Indeed, if anything, the use of milk in westerns can reduce specific signal (probably by the small amounts of free biotin in the milk blocking the streptavidin binding sites).
I have produced good blots by using milk in the blocking and primary antibody step. Then washing extensively with TBS+tween (to remove free biotin) prior to incubating with the strepavidin conjugate in TBS+BSA.

With streptavidin it can be a good idea to 1) include Tween in all solutions 2) use a slightly higher ionic strength (200-300 mM NaCl) in the conjugate incubation buffer 3) include some carrier such as BSA or casein in the conjugate buffer.

Always find the optimal dilution of each new batch of your streptavidin conjugate. Run a biotinylated protein in every lane of a gel. Do the transfer, dry the membrane then cut it into strips with a scalpel. Block the individual strips, and incubate individual ones with dilutions of your conjugate.

Hope this helps.

#3 biologist2B

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Posted 13 April 2009 - 03:35 PM

Thank you so much! That was super helpful!

What biotinylated protein do you typically use? I can use that in my initial studies to find out the optimal dilution of of my streptavidin HRP. I can also use that as a positive control as well.

Thank you so much! You're like the postdoc our lab needs!

#4 klinmed

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Posted 15 April 2009 - 10:28 AM

Thank you so much! That was super helpful!

What biotinylated protein do you typically use? I can use that in my initial studies to find out the optimal dilution of of my streptavidin HRP. I can also use that as a positive control as well.

Thank you so much! You're like the postdoc our lab needs!

Glad to help. This is what the forum is all about.

I usually use biotinylated mouse IgG which we make in house (for other purposes). Any biotin-labelled protein will do. Preferably one you have in the lab already or can get free from a colleague.

If you use a protein about the size of the one you want to detect it will also be a control for transfer efficiency.

Remember that you need to load very little on the gel. The amount depends on your light-generating reagents (see manufacturers literature). It is usually a good idea to dilute it in buffer containing some carrier protein (eg 200 ug/ml BSA).

Hope this helps




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