General questions about western - (Dec/21/2005 )
Hi, several general questions,
1. BLOCKING : Someone said that using 3~5 % BSA istead of skimmed milk as blocking buffer or Ab diluent can enhance the signal more but reduce specificity. Did anyone have such experience as mentioned ?
2. WASHING : Who plays the more important role that determines the cleaning effect in TBST washing buffer ? NaCl or Tween 20 ? If I want to get a clearer background, whose concentration should I adjust ?
3. TRANSFER : I ran 6% gel and transfer a target protein with 130KDa onto membrane(wet transfer) , is it necessary to use transfer buffer which contains SDS ? I did add 0.0375 % SDS but the volt reduced a lot and tranferring was bad (Ponceau S check). Does SDS increase the "electric resistance" ?
looking forward to your responses
BSA does increase background in westerns. Blocking with 5% milk in TTBS is the way to go. However, some specific companies who make phosphate-specific antibodies ask for the blocking to be done in BSA. Personaly, i hate bsa..milk works better for me.
Tween-20 plays the more imp role of reducing non-specific binding and background. Tweek the Tween 20 concentration (0..1%-0.5% is the usual range with the 0.1% being the most common.
SDS is not a part of transfer buffer. Methanol is the most crucial thing in TB. normally TB comprises of 20% methanol . The other ingredients are tris-base and glyceine in water.
I would like to add, milk is awesome for most applications, but you must beware if you are using a streptavidin system; milk usually has some endogenous biotin that will interfere and crank up your background like no-one's business
with applications using streptavidin, I usually block with 5% BSA in standard PBS/tween, then add 1-3% BSA to the PBS/tween for the washes and the antibody dilutions. I am sure that TBST would work the same way here....Otherwise we always use milk, it's cheap and easy and works great
SF, another thing, if you are having too much background, do you mean too many bands or too much signal across the membrane? if it is 'b', then I would also recommend trying a bit less secondary and washing a bit longer