Blocking buffer recipe - (Jul/08/2008 )
Hey everyone!
I've always used BSA in my blocking buffer--- however, now I'd like to switch to use non-fat dry milk in Tbs-T. What percentage of non-fat dry milk would be good to use??
Thanks.
5%milk in TBST works the best
Yeah...5% skimmed milk should do the work
Wowie is right. 5% is what's commonly recommended. If you want a cleaner blocking buffer you can use an ultra-centrifuge...
5 g non-fat dry milk
100 ml TPS (most people use PBS but both work great)
-dissolve non-fat dry milk in buffer
-adjust pH to 7.4 (or what ever you need it to be)
-In ultra-centrifuge spin at 100,000 g for 1 hr
- collect supernatant and filter through a standard 0.2 micron filter
Store at 4 degree C
Do this and your buffer will be a nice clear liquid but still have a high concentration of Cassein, which is the protein in milk that does the actual blocking... Note also, that this means your buffer is sterile as well. Most people don't bother to filter the buffer after dissolving the milk in it, likely because it's very difficult to do. If you don't use an ultra-centrifuge first the solution is too thick to get through most filters; it absolutly will NOT go through a 0.2 micron filter.
However, if sterility isn't a big issue, then don't waste the time with the ultra-centrifuge and what not.
I use 5%FBS in PBST(.05% tween20) the ph of PBS is 7.40(+-0.1)
My blocking buffer always makes freshly
FBS is from -80 freezer.
5 g non-fat dry milk
100 ml TPS (most people use PBS but both work great)
-dissolve non-fat dry milk in buffer
-adjust pH to 7.4 (or what ever you need it to be)
-In ultra-centrifuge spin at 100,000 g for 1 hr
- collect supernatant and filter through a standard 0.2 micron filter
Store at 4 degree C
Do this and your buffer will be a nice clear liquid but still have a high concentration of Cassein, which is the protein in milk that does the actual blocking... Note also, that this means your buffer is sterile as well. Most people don't bother to filter the buffer after dissolving the milk in it, likely because it's very difficult to do. If you don't use an ultra-centrifuge first the solution is too thick to get through most filters; it absolutly will NOT go through a 0.2 micron filter.
However, if sterility isn't a big issue, then don't waste the time with the ultra-centrifuge and what not.
I use 0.45 filter and seems to be ok....I make 5%BSA buffer in PBS pH7.4.....I read somewhere that when we filter our pH increases, so you might need to double check your pH.