Author Topic: Many waves in my gel!!??  (Read 12116 times)

moA

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Many waves in my gel!!??
« on: January 02, 2007, 05:00:00 PM »
My sample is E. coli BL21 cultured in LB and washed with low salt washing buffer 4 times.Pellet was lyse with 8M urea, 4% CHAPS, 40mM DTT(standard for 30min and then sonicate 40s).Centrifugation at 22000 rcf, 15 degreeC. Mix about 4ul(100ug) of the extracted protein with 336ul rehydration buffer(8M urea, 2% CHAPS, 20mM DTT and 0.5% Bio-Lyte).
Active rehydrate for 16h.IEF was performed as 500v for 30min, 500-4000V for 1.5h, 4000V for 24000VHr and finally freezed at -80 degreeC overnight.
SDS-PAGE condition is 10mA per gel for 1h and 40mA per gel to the end.
It was a 12.5% gel.
Stained by silver nitrate.

The attachment is the result. There are many waves especially the high molecular weight region.Is this caused by casting gels unevenly? or it's overfocusing?
There are some vertical smear in the top of the gel, what's that?
I need your comments, many thanks.

regards
moA


Reiner Westermeier

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Re: Many waves in my gel!!?? (moA)
« Reply #1 on: January 02, 2007, 05:00:00 PM »
Your gel shows a depletion of glycine in the second dimension. Could it be, that your upper buffer was too diluted or the volume was too small?

moA

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Re: Many waves in my gel!!?? (Reiner Westermeier)
« Reply #2 on: January 02, 2007, 05:00:00 PM »
Reiner, do you mean the depletion of glycine caused the wave or the smear in the  top region?

regards
moA


Reiner Westermeier

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Re: Many waves in my gel!!?? (moA)
« Reply #3 on: January 03, 2007, 05:00:00 PM »
Yes, you see the typical horizontal line, above which you see only blurred spots and smears? This comes from glycine depletion.

moA

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Re: Many waves in my gel!!?? (Reiner Westermeier)
« Reply #4 on: January 03, 2007, 05:00:00 PM »
Thank you, Reiner.
I used Bio-Rad system(but GE strip) and always use the same buffer in the top and down tank. But the others in my lab operating other samples(tissue or cell), this phenomena did not occur. This also occurred just a few times in my experiments.

regards


moA

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Re: Many waves in my gel!!?? (Reiner Westermeier)
« Reply #5 on: January 04, 2007, 05:00:00 PM »
So I have to increase the conc. of the upper running buffer to 2X? Or just increase glycine conc.?

regards
moA


moA

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Re: Many waves in my gel!!?? (moA)
« Reply #6 on: January 07, 2007, 05:00:00 PM »
Here's the new one I done just yesterday. The sample is also E. coli under different condition.
I increased the voltage to 4500 in the step 2 and Vhr to 27000 in the step3. I also increased the upper running buffer to 2X.
The result is bad, anyone can give commons?
Thank you!!

Reiner Westermeier

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Re: Many waves in my gel!!?? (moA)
« Reply #7 on: January 09, 2007, 05:00:00 PM »
It seems that the glycine problem is solved now.
I cannot judge on the first dimension, because i have no info on strip length and pH gradient. furthermore I cannot judge on biolytes, I just know that IPG bufffers and Pharmalytes from GE work very well.
And something is wrong with the second dimension gel. It seems that overlaying was not very good..

moA

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Re: Many waves in my gel!!?? (Reiner Westermeier)
« Reply #8 on: January 09, 2007, 05:00:00 PM »
Sorry, I forget it. It is an 18 cm pH 3-10 NL strip and the left side is pH3.Reiner, you mean the agarose overlaying is not flat?

moA


Reiner Westermeier

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Re: Many waves in my gel!!?? (moA)
« Reply #9 on: January 11, 2007, 05:00:00 PM »
No, overlaying the gel after casting.

moA

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Re: Many waves in my gel!!?? (Reiner Westermeier)
« Reply #10 on: January 15, 2007, 05:00:00 PM »
Overlaying looks good. Is 30KVh enough for a 18cm pH3-10 NL strip? I found there are a great range from 30 to 80KVh in papers I have read.

regards
moA


Sjouke Hoving

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Re: Many waves in my gel!!?? (moA)
« Reply #11 on: January 15, 2007, 05:00:00 PM »
30 kVh is a little bit on the low limit - you can easily go to 50 kVh with this one. But actually, the IEF part doesn't look too bad. It is something in the 2nd dimension. To me it looks like bad polymerization of the gel. How fast is polymerization?

sjouke


moA

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Re: Many waves in my gel!!?? (sjouke)
« Reply #12 on: January 15, 2007, 05:00:00 PM »
I did not notice the time it polymerized but the remained solution in the beaker start to polymerize after more than 20min.
Sjouke, do you know why the left-top(acid-high MW) region is with a darker background? It is because there are many proteins in this region?
Many thanks!

moA


Sjouke Hoving

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Re: Many waves in my gel!!?? (moA)
« Reply #13 on: January 17, 2007, 05:00:00 PM »
That darker area contains many proteins, and especially there the focusing is not optimal (maybe a bit overloaded).
I would suggest to use 7M urea / 2M thiourea and Pharmalytes instead of Biolytes.
But the other thing is more the 2nd dimension for which I really don't have a good explanation yet. How many gels are you casting at the time? Which 2nd dimension system you use?

sjouke


moA

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Re: Many waves in my gel!!?? (sjouke)
« Reply #14 on: January 17, 2007, 05:00:00 PM »
Thank you, sjouke. I usually cast two gels at the same time but I cast only one in that experiment.

moA