Funneling Westerns

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Laazie83
Laazie83's picture
Funneling Westerns

Hi,
I'm using trying to get rat white fat samples to run properly on my gel. 120V, max amps is what I'm using now. My problem is that every second loaded sample is running into a funnel...but not all of them. Should I drop the voltage? Do you think there is protein aggregation (if so, what would you recommend?) My protein's around 31kDa. I started from scratch with the extractions, thinking something had happened there, but now these newly extracted samples are funneling more often than the last ones! (Still not every one though). Any ideas? By the way, I don't pour my own gels. I use the NuPage system.
Thanks!

SanDiablo
SanDiablo's picture
Without knowing the specifics

Without knowing the specifics of your experimental conditions, this sounds like a salt or solvent problem. I've seen issues like this when I alternate conditions in my sample loading, eg extractd, unextracted, extracted. If something like this is the case, try running samples prepped by similar methods next to each other, with an empty well in between the preparation methods. This way, salt or solvent variations between samples are not interfering locally with each other so much.

Otherwise, some additional sample clean-up may be in order...

Working with fat is yucky!

Laazie83
Laazie83's picture
:-)

:-)
Thanks for the advice. The preparations of the samples has been exactly the same for all of them though, so they shouldn't be interfering. As far as "additional sample cleanup" goes, is there anything special you'd advise? Also, could I be running the gel at too high a voltage perhaps? They all funnel when I run it at 150V. Now I'm running it at 120V (which wasn't a problem before, but Westerns being temperamental at the best of times, perhaps I need to lower it further?) (It could also be a problem with the Laemmli or the RIPA sample buffer I guess). Would spinning the samples down quite vigorously for a short period maybe break up the proteins (UCP-2) if they're in aggregates and that's why there is funneling? Or would that just result in me losing a significant amount of my protein as a pellet? Just throwing around ideas. :-)

Paul Weinreb
Paul Weinreb's picture
Are the samples viscous when

Are the samples viscous when you're loading them? Often with certain cell extracts, for example, you tend to get "goop" that can disrupt the running of the gel. The solution to this is generally just to boil the samples for several minutes in SDS-containing sample buffer - are you boiling the samples for ~5 minutes before loading? (my guess is yes)

It doesn't sound like the problem is the protein; usually if you've got aggregates they just stick up at the top of the lane, and the soluble proteins run OK.

What's the extraction buffer? RIPA? Any unusually high salt concentrations (>500 mM)?

PW

Laazie83
Laazie83's picture
The samples don't seem

The samples don't seem viscous, but I will try boiling for longer. Yes, my extraction buffer is RIPA and I don't think I have any really high salt concs. I'll only be re-running next week, but I'll let you know if it helped.
Thanks!

vanishing
vanishing's picture
funneling is usually a sign

funneling is usually a sign that you have overloaded the lane
have you tried loading less sample?

dawn
dawn's picture
If this helps we were

If this helps we were routinely running protein from adipose tissue using the NuPage system. We would boil the sample for 10 minutes and run at 200 volts.

vanishing
vanishing's picture
so were you able to solve the

so were you able to solve the problem?
and if so, what worked?