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Protein SDS-PAGE thick bands / smear


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#1 TunaMelt

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Posted 22 October 2010 - 04:24 PM

I've always had wide bands for these proteins and I believe there is a way to sharpen them. I believe the problem is probably the sample buffer formula. I've scoured these forums and tested a few suggestions: add NaCl, add Urea, add MgCl2, decrease loading size (just got fainter, not narrower). But nothing helped.
Attached is an example of a cropped gel. The line going from left to bottom right is an artifact of scanning the gel.
lanes with smeared protein are what I'm working on, crisp lanes are standards.
10% gel, Coomassie stained.

In an alternate problem, there is the outline of the lane that is a light smear. I've read that decreasing salt might fix that; corroboration? and this Sample buffer doesn't have NaCl added to it, what should I decrease?

Sample buffer formula:
33% glycerol
100mM DTT
10mM EDTA
10mM Tris-HCl
3% SDS
0.004% Bromophenol Blue
pH 7.0

Attached Thumbnails

  • 5062:84 10%.jpg


#2 donny

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Posted 23 October 2010 - 04:08 AM

What is the recipe of your stacking gel? Are you using enough stacking gel? The protein concentrates to a fine band while in the stacking gel so it's important to have a reasonable height of it. Could there be DNA contamination? Genomic DNA may cause smears. I'm not sure if 0.2 pH units make a huge difference but most loading dye recipes are at pH 6.8, where glycine is uncharged. Anyway, I think you are loading too much protein in your gel.

Edited by donny, 23 October 2010 - 04:13 AM.


#3 mdfenko

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Posted 25 October 2010 - 07:17 AM

to continue with donny's comments about stacking gel, the stacking gel should be at least 5mm from the bottom of the comb teeth to the running gel for a minigel and 1cm for a standard gel for proper stacking.

that pH difference is a concern, it should be 6.8 for the laemmli buffer system (at least for the stacking gel buffer).

more protein appears to be loaded than necessary. how much did you load?

salt is contributed by the buffer that the sample is in before adding the loading buffer.

addition of urea can sharpen the bands. add it to the gel formula, not just the sample buffer.

you can also sharpen the bands by using a gradient gel.
talent does what it can
genius does what it must
i do what i get paid to do

#4 TunaMelt

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Posted 26 October 2010 - 09:16 AM

first, thanks for the reply. This is the best community of support I've ever seen for everything biology.

stacking gel recipe:
4% Acrylamide
0.1% Methylenebisacrylamide
0.125M TRIS
0.1% SDS
pH 6.8

I thought the gel or running buffer wouldn't be the issue because the standard looks just fine.
I'll try making new sample buffer at pH 6.8
no chance of DNA contamination - these are purchased pure proteins.

currently loading 4g per well
I ran a range of proteins (0.8-4g/well), and lower protein amounts with pH 6.8 do give smaller band (but pH 7.0 doesn't). I think that's what I was missing the last time I tried decreasing the amount. Looks like I might need to decrease the amount of protein in each well even more. I will try a 25-400ng/well range.

#5 TunaMelt

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Posted 27 October 2010 - 09:18 AM

oh yeah; this is the solution to what I came here to fix.
lane 1 = 40 ng
lane 2 = 80 ng
...
lane 15 = 500 ng

lane 5-10 is exactly what I'm looking for (200-400ng/well)
thanks for the help!

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