Buffers - Confused about the compositions (Jun/03/2009 )

Hi peeps,

I am going to design an experiment, and I need to come up with blocking buffers. I am new to this so do pardon me if I sound stupid!

I have heard of people using tween in PBST and milk in western blot blocking buffers to prevent non-specific binding. However, how does all these blocking substances (tween or milk for example) work? How should one determine their concentration in the whole buffer? And how do we know what other ingredients can be used to prevent non-specific binding or for more stringent washings?

Thanks in advance!

jiajia1987 on Jun 3 2009, 10:45 AM said:

tween will help to wash out proteins that bound non specifically (weak binding). Milk contains protein (you can also use BSA) that will bind via protein protein interactions, non specifically. Then you will have a competition between milk proteins and your antibody with the non specific binding sites. There is a competition for the specific sites, too, but the antibody will win.
As there is an excess of the blocking proteins, you will mainly have milk proteins bound on non specific sites and antibodies on specific sites.
Usually we use up to 5% non fat dry milk or 1% BSA.
You have to try which one is the best, and try different concentrations, increasing if you have background, decreasing if you don't have enough signal.

little mouse on Jun 3 2009, 05:23 PM said:

Dear little mouse,

Thanks for explaining to me.

Other than tween and milk, what other things can we use to wash non-specific binding proteins or used for blocking? it may not necessary be for western blot. I am actually doing an experiment whereby I have streptavidin-coated beads and I have biotinylated anti-HA antibodies bound onto it. At the same time, I have biotinylated templates (with a HA sequence at the 3' end) bound onto the beads too. WIth in vitro translation, proteins (with HA tags at the end) are produced and can bind to the biotinylated anti-HA antibodies on the beads. I am using this peptide conjugated to FITC to detect the proteins, but there is a sign that the peptides are binding nonspecifically to the beads.

The solution would be to have a more stringent washing during the washing steps, or include a blocking buffer during the incubation steps where I am incubating the antibodies and the templates with the beads. Alternatively, I can get a more specific-binding peptide, but that is all I have now and my supervisor would prefer to try out a more stringent washing or a blocking buffer. Is there a website where it carries information on buffers that can be used for such purposes?

Many thanks.

i am sorry to add on. in addition, when do we know when the buffers should be filtered-sterilized?

Should it be filter sterilized ? the washing buffer and all that stuff , i made them up and don't even bother sterilizing.

hanming86 on Jun 4 2009, 11:47 AM said:

Okay thanks a lot!

you can increase the NaCl concentration up to 0.5M to reduce the binding of non biotinylated proteins. Some time it helps.

jiajia1987 on Jun 4 2009, 03:24 AM said:

I would block with BSA, not milk, and I would increase the concentration of NaCl up to 0.5M during the washing step.

Hi little mouse,

Thanks for your reply.

Why would BSA be a better choice than milk?

And where does NaCl come into the washing steps? So far, my washing buffer contains 0.1% (w/v) BSA, 0.1% (v/v) Tween-20 in DPBS. Does NaCl come in the form of a powder? I am sorry if this sounds stupid.

jiajia1987 on Jun 4 2009, 11:37 AM said:

no milk because there is biotin in milk.
DPBS is Dulbecco's PBS. PBS is phosphate buffer saline, with 150 mM NaCl
Buy some NaCl powder from sigma and add it to your solution.