help optimize an western blot for 6kd protein - western blot tranfer problem (Mar/10/2008 )
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
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?
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!
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?
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
q.s. to 1000ml with distilled H2O , I use Ix of this and 200ml of methanol.
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