Tricine-SDS gels blotting problems - (May/02/2011 )

Dear community,

I need your experience with Tricine-SDS gels (Schaegger gels).
I want to detect an 11 kDa protein in a western and decided therefore to run a tricine-SDS gel.
The gel casting and run itself is fine in my eyes. But the problem arises after the blotting.
I blot semi-dry with a modified 3 Buffer System (Kyhse-Anderson, 1984)

Cathode buffer:
525 mg epsilon-Aminocaproinacid in 100 ml Anode buffer I
Anode buffer I:
25 mM Tris-HCl pH 10.4 , 20% (v/v) Isopropanol
Anode buffer II:
300 mM Tris-HCl pH 10.4 , 20% (v/v) Isopropanol

for 1 h at 0.8 mA/cm2.

This system is fine with glycin-SDS gels and so I assumed it will also work with tricine-SDS gels.
But after staining the membrane (Nitrocellulose 0,45 µm) with Ponceau (0,2% in 3% TCA) I can
hardly detect stained bands and staining of the blotted gel with Coomassie shows me a bunch of bands
still in the gel (also the marker is not blotted completely – I load 5 µl of Fermentas
Prestained Page Ruler.

Has anybody an idea what’s the mistake is, I am not aware of?

Thanks in advance!

PS.:
To be sure I prepared all buffers new and checked the pH carefully. I also used two different
blotting machines, to check if the electrodes are to old, but still the same result –
Glycin-gels are blotted well, but not the Tricine-gels.
I use 49,5T/3C for Tricine-SDS gels and 37,5T/1C for Glycine-SDS gels, is this the striking point?

do you equilibrate the gel in transfer buffer before blotting?

if not then you have two factors slowing transfer, tricine and more extensive crosslinking.

if so, then more extensive crosslinking is most likely your problem.

you can increase transfer time and or current density to improve the result.

also, i use a similar buffer system for semidry transfer but i adjust the pH of the cathode buffer to 9.4 and use methanol, not isopropanol.

I got the best results using Tris-TRICINE- SDS PAGE (12% gel 29:1 acrylamide:bis)and after that semi-dry transfer onto a PVDF with 0,2um pore size (bigger pores result in blow through of small proteins like yours). I use a standard TRIS-glycine-methanol buffer (25mM Tris, 192mM Glycine, 10% methanol) and 0.8mA/sqcm of transfer. In my case (a 10kD protein), it worked wonders. You can also try to drop methanol from the transfer buffer if you keep having problems.

Good luck!
K.

kristoff on Tue May 3 20:49:25 2011 said:

do not remove methanol from the transfer buffer. it is required to strip sds from the protein so that it won't interfere with binding to the membrane. you can adjust the concentration (in the range of 10-20%) if it is interfering.

mdfenko on Mon May 2 16:44:19 2011 said:

Thanks for your replies mdfenko and kristoff!

I normally agitate my gel in transfer buffer very shortly - I don't want to loose sample by diffusion.

So I wasn't aware of the necessity to equilibrate the gel, to remove the Tricine.

This leads me to the question, why is glycin added to Western transfer buffer when at the same time I want to remove it out of the gel?
(I know the function of Tricine/Glycin in isotacho-electrophoresis, but the function for Glycin in the blotting buffer is nebulous - has anybody a flashlight for me ?)

@ mdfenko: Why is Tricine slowing the transfer?

I will try a 10%T/3%C gel instead of 15%T/3%C and also equilibrate the gel longer in transfer buffer before blotting, maybe this and
blotting with 1 mA/cm² will help.

Thanks!