Western Blotting protocol-specific questions - (Jan/24/2006 )

I have questions regarding the different protocols for western blot.

For the transfer buffer:
1. Does anyone use isopropanol instead of methanol? Any advantages using isopropanol?
2. Does anyone use anything else other than 25mM Tris and 192mM Glycine? Any other concentrations of these components? What is the reason for changing these?
3. Does anyone use NuPAGE Bis-Tris gels? What is the composition of the transfer buffer you use?

For blocking buffer:
1. Does anyone adjust the pH of the blocking buffer after addition of the dry milk?
2. Does anyone prepare the blocking buffer as follows:
-measure out the volume of TBST
-measure out the grams of milk
-pour the dry milk on top of the TBST and DO NOT mix. Allow the milk to dissolve slowly into the buffer --> this takes a super long time.
-centrifuge down the undisolved milk and use the supernatant for blocking
-this procedure claims to lower the background on western blots

---

about blocking buffer with milk :

I weight the desired quantity of milk.
I add the desired volume of TBS (I do not use tween for blocking).
I vortex. and put it directly on the membrane.
it happened some time that there was an aggregate of milk. don't care.
incubation overnight at 4°C (because I do not want to spend my night in the lab) and I have no background.

---

Greetings!

About the blocking buffer- isn't this technique the same as using less powdered milk in the first place, but slower to mix?

monkeybaji

---

The way I understand it (have in mind that this is the first time I also hear about this technique) is that dissolving milk in TBS-T creates sort of a suspension, i.e. not all particles are soluble in the TBS-T, this technique allows for the removal of such particles (at least to some extent). I am unsure as to what these particles may be, they could be the more hydrophobic proteins (or trace lipids?). The question is does it really matter if you remove this stuff from the milk?

---