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Blurry bands ...


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#1 Drake1982

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Posted 07 July 2012 - 06:23 AM

Hello,
I've been running SDS Pages for a while ... lately I was doing some DNA affinity chromatography experiments and was running the elutions on a 16% gel. My particular interest is the identification of smaller proteins (around 10 - 20 kDa). I don't know why, but apparently the smaller proteins on my gel, appear as blurry bands (see attached file); thus giving really troubles for further MALDI analysis.

Does anyone know how I can compress the lower bands? Would it make sense to run a 10% gel and just stopping the run earlier? Why does this actually happen?

Thanks in advance,
Cheers, Daniel

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  • DSCF1259a.jpg


#2 bob1

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Posted 07 July 2012 - 10:26 AM

I would check the salt content of the elution buffer. You could try dialysing against a lower salt buffer to bring the salt down a bit.

#3 Drake1982

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Posted 07 July 2012 - 11:19 AM

Hi,
yeah .. I thought about this issue already before ... the thing is, with a DNA affinity chromatography, you have to increase the salt concentration in the elution buffer ... dialysis would be an option .. but would prefer an easier way ...

as for the attached image (from left to right) from 100 mM NaCl to 1 M NaCl.
As you can see, the bands I'm interested in, are from 100 mM to 500 mM NaCl (with the strongest band around 350, 500 mM) .. actually I was assuming that this salt concentrations would not interfere with the run ...

... would you have any suggestions on how to concentrate the elution? In a sense, how would you do a dialysis when you only have about 40-50 ul of elution ...

Thanks!

#4 Adrian K

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Posted 07 July 2012 - 09:24 PM

Hi Drake,

I suggest you might need to look into your buffer's voltage and temperature especially after a log run. Also, perhaps you might need to change by using new APS and B-mercaptoethanol. I used to have blurry bands when such things were not right.
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#5 Drake1982

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Posted 07 July 2012 - 10:03 PM

Hi Drake,

I suggest you might need to look into your buffer's voltage and temperature especially after a log run. Also, perhaps you might need to change by using new APS and B-mercaptoethanol. I used to have blurry bands when such things were not right.

Hi,
Thanks, I might give this a shot ... but since I'm not the only one using the solutions, and others don't have this problem, it might not cause the problem. Furthermore, when you look at my gel, you see that the higher bands (starting from around 50 kDa) do not appear to be blurry ... only the lower ones ...

#6 proteaMatt

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Posted 09 July 2012 - 04:41 AM

I like to use 3kDa amicon filters when I run serum samples, those would probably work well with your application. I have used those filters on samples anywhere from 50 uL to 400 uL.

Also, I can't really tell from the picture you posted, but are you using a stacking gel? That can definitely help to increase resolution.
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#7 Drake1982

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Posted 09 July 2012 - 08:28 AM

I like to use 3kDa amicon filters when I run serum samples, those would probably work well with your application. I have used those filters on samples anywhere from 50 uL to 400 uL.

Also, I can't really tell from the picture you posted, but are you using a stacking gel? That can definitely help to increase resolution.


Hi, Thanks for your reply! The idea with the filter sounds great .. hope they are not too expensive. ... And yeah, I do have a stacking gel, but cut it away before I started with the staining ...

#8 mdfenko

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Posted 09 July 2012 - 08:30 AM

a couple of things:

you can desalt by drop dialysis

resolution of low mw proteins and peptides is often poor, even with a high acrylamide percentage, with tris-glycine-sds gels. you can get exceptional resolution of your proteins of interest with a 10% tris-tricine-sds gel (shagger and von jagow).
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#9 Drake1982

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Posted 09 July 2012 - 10:45 AM

a couple of things:

you can desalt by drop dialysis

resolution of low mw proteins and peptides is often poor, even with a high acrylamide percentage, with tris-glycine-sds gels. you can get exceptional resolution of your proteins of interest with a 10% tris-tricine-sds gel (shagger and von jagow).

Hi, Thanks for your answer .. I might give this a shot tomorrow .. is there any chance you have a protocol available; for preparing the gel as well as the buffer? Can I run those gels with any system? Since I found this statement:

Tris-Tricine-SDS (TTS) running buffer is the cathode (upper reservoir) buffer

I'm a little bit confused .. what does it mean with the upper reservoir? Do I need a special system for that .. do I also need an anode buffer?

Thanks, Daniel

EDIT: So I guess the upper reservoir is the part of the assembly between the glass plates and the lower reservoir is whole tank?

Edited by Drake1982, 09 July 2012 - 11:42 AM.


#10 mdfenko

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Posted 11 July 2012 - 07:21 AM

here's the protocol (in rich text format):

Attached Files


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#11 Drake1982

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Posted 11 July 2012 - 08:41 AM

here's the protocol (in rich text format):


Thanks for the protocol .. I've already found a similar one online and already tried the run yesterday ...
but unfortunately, it totally failed ... I completely overlooked the fact that you run it at low voltage for quite a few hours. So after realizing that my running front was basically not moving, I increased the voltage ... which in fact was not a good idea ... so I ended up with a totally screwed gel .. not telling me anything ... but I'll repeat it with an overnight run at low voltage

... do I have to cool the buffer or can I run it at room temperature overnight?

BTW: How can I understand your gel preparation? What are the 10% spacer and the 10% running good for? And do I need them? And what would the size
be? And what do you mean with (runs “–“ to “+”)?


Thanks!

Edited by Drake1982, 11 July 2012 - 08:51 AM.


#12 mdfenko

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Posted 11 July 2012 - 10:20 AM

it is run at room temperature.

the original formulation had a 3 part gel: stack, spacer, and running gel. the 10% running gel is good for separating proteins in your size range. the 16.5% running gel is good for smaller proteins and peptides (we used it for neuropeptides including enkephalins).

we found that we could omit the spacer gel if we run a gradient gel.

if you look at the original paper, it gives the length that they worked out for each layer (you can scale them to your dimensions).

runs "-" to "+" is to tell the order of the electrodes (this is the standard configuration).

here is a later version of the protocol (with explanations):

Attached Files


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#13 Drake1982

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Posted 11 July 2012 - 10:44 AM

That's really nice! Thanks a lot! I appreciate your help!




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