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help optimize an western blot for 6kd protein - western blot tranfer problem (Mar/10/2008 )

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I am currently usind 15%glycine SDS page to run a western.. verything is fine until i transfer the protein into the blot ,,, after overnight incubation i dont see my protein of interest but i see the all the upper bands.. my trancer buffer is tris, glycine and 20 methanol and i transfer for 2 hrs at 100V. It used to work fine, but now does not

today i run the gel again and protein got stuck on the top of the gell even through i boiled at 100C for 5mins
plz help mellow.gif

-mama wa nyumbani-

you could try to use a gradient gel, perhaps 4-16 or 10-20% would help to display your protein better.


100V seems pretty high for a transfer. I usually transfer 15% mini-gels at constant current - 250mA for 45 minutes.

I think this is the calculation for how many amps to use for transferring.
Amp = surface area of membrane (in cm2) X2


I would agree that 100V for transfering sounds like a lot. I apply 25-30 Volts for 2 hours and have never had problem. I am also working with a small protein (10.5 kDa) and I have used homogenous 10% and gradient 10-20% gels, but I admit that it did not make a huge difference! Hope everything goes better for you!



hi i was wondering what kind of gel r you using tris-tricine or tris glycine and what percentage ? i was i advice to use 13.5 gel and will i still transfer the same time?

-mama wa nyumbani-

QUOTE (mama wa nyumbani @ Mar 12 2008, 08:29 AM)
hi i was wondering what kind of gel r you using tris-tricine or tris glycine and what percentage ? i was i advice to use 13.5 gel and will i still transfer the same time?

HI. I'm also doing a western on a small protein (about 12kda). I've been doing 12-16% tris-tricine gels, with PVDF membranes, and 20% MeOH/glycine transfer (wet for 45 min at 100v). I've been getting somewhat better results than previously. I'm also going to trying a CAPS transfer buffer to see if I can get a better transfer. I'll let you know the results.



I also regularly do western blots for a 12kDa protein, and i have never once had any trouble. i make 12% tricine gels (although i am sure you could go ab bit higher than this for a 6kDa).

here is my transfer buffer for the western blotting:
39mM Glycine, 48mM Tris, 0.037% (w/v) SDS, 20% (v/v) Methanol, pH8.3 with HCl.

for a standard thickness mini gel (0.75mm) i blot for 18 mins at 13v on a biorad semi-dry.

my gels andf westerns are always perfect!

-Flour Power-

although it also sounds like you are not running your gels properly, as the protein is getting stuck. what are your recipes and running conditions?

-Flour Power-

i useally load about 40ug/lane and add sample buffer (b-merc) which it stored in -20C, I then mix by vortex and boil the sample
for 5mins and load into a wells. the recipe for running buffer is

30 g of Tris base
144.0 g of glycine
add 10 g of SDS
dilute to 1L of distilled water - i onlu use IX, i usually never adjust the pH. i let it run for 50V until it passes the stacking gell and the 100V until 3/4 of the gel because my protein is small and i dont want to loose it so i dont let the dye run out. I transfer ( wet) for 100V for Ihr in the cold roomAdd the following to 800ml H2O
36.35g Tris
150g Glycine
4g SDS
q.s. to 1000ml with distilled H2O , I use Ix of this and 200ml of methanol.

-mama wa nyumbani-

i know its quite hard to make tricine gels in my lab cause we dont have the degassing apparatus, thats why i use the tris, glycine gels, and we do wet transfers,s o can i still use the same buffer and time you suggested?
-- I wonder if you can cast the trice gels as glycine gels .. can you ? plz let me know

-mama wa nyumbani-

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