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No protein bands in SDS-PAGE after sample clean-up steps! HELP!


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#1 rachelhauser

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Posted 26 January 2010 - 04:02 PM

Hello everyone, I'm new to the forum, and am looking for some help...

I am doing a proteomic study on animal fluids that, at the beginning of my study presented some well-defined protein bands on a 10% SDS-Page (1D - MW separation only), using coomassie blue.

When I started my clean up (removing lipids and ac/Et-OH protein precipitation, desalting columns - Midi-Trap G-25 from GE - Albumin and IgG removal kit from GE), I noticed NO BANDS appeared in my 10% gels...

I ran the samples on gels after each clean-up step, and have tried using samples with and without SB buffers, and the results are the same, I only obtained very two faint protein bands when using the desalting columns.

The gels are fine, no problems there, the MW standards run fine, the system is fine, solutions are fine... I really need help on the sample preparation... can anyone help?

The samples are highly complex fluids, and need a desalting step. The lipid removal may not be necessary... but I am at a loss for the lack of proteins in the precipitated protein sample, since I though this method was supposed to precipitate ALL PROTEINS, and concentrate them, and I am following a paper on the method EXACTLY, to the letter, regarding the same fluids as I am working with (though the authors only quantified the proteins, did not do SDS-PAGE with them).

I am thinking of running a zymograph to see if maybe I can see any proteins with activity... but that doesn't make sense right? If I see something in the zymograph it should also be apparent on a coomassie Blue stained gel, right? The only thing I can't see is the presence or absence of activity on a normal SDS-PAGE gel, but the protein should still BE THERE, right?

I will also be staining the gels with silver, which is more sensitive, to see if I can see anything in the samples... But if anyone can help I'd very much appreciate it, with any suggestions. And maybe do the Zymograph, if no one eles can shed a light on anything...

Sorry about the length of this post...

Rachel
"Live Long and Prosper".

#2 K.B.

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Posted 27 January 2010 - 04:56 AM

Do you know how much protein is there in this fluid? Perhaps you don't see any precipitate because there's so little of it?

#3 rachelhauser

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Posted 27 January 2010 - 06:29 AM

Do you know how much protein is there in this fluid? Perhaps you don't see any precipitate because there's so little of it?


Hello, thank you for trying to help :D I appreciate it!

Well... I quantified proteins by 2D Quant Kit (GE), and obtained that in 10 uL of fluid, there is a mass of approximately 90 ug of protein... when I loaded the samples on the gel, I used 60 - 80 uL of the same sample... that would give an approximate protein count of 540 - 720 ug per lane... That would be enough to stain at least somewhat on a 10 % regular gel right?

:)
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#4 mdfenko

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Posted 27 January 2010 - 07:42 AM

yes. that should stain very well.

try silver staining, as you point out it is more sensitive than coomassie.

it is not clear from your response if you determine the protein concentration after each step in your protocol. you should be doing this, especially since you haven't followed this protocol in the past.

also, keep in mind that many authors omit some specifics in their procedures (unintentionally as well as intentionally).

must you perform the precipitation step? you may be losing too much protein with this step. try omitting it. by the way, is it ac/etoh or tca/etoh?

Edited by mdfenko, 27 January 2010 - 07:44 AM.

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genius does what it must
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#5 rachelhauser

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Posted 27 January 2010 - 07:58 AM

yes. that should stain very well.

try silver staining, as you point out it is more sensitive than coomassie.

it is not clear from your response if you determine the protein concentration after each step in your protocol. you should be doing this, especially since you haven't followed this protocol in the past.

also, keep in mind that many authors omit some specifics in their procedures (unintentionally as well as intentionally).

must you perform the precipitation step? you may be losing too much protein with this step. try omitting it. by the way, is it ac/etoh or tca/etoh?


Hello, thank you for your help... I have determined the protein concentration after each step yes, an strangely enough, after each step I lose only a few ug of protein! After delipidation, approximately 100 ug of protein --> after precipitation --> approx. 90 ug of protein! Thatīs the strangest part of it all... I should still be able to see something after precipitation, not NOTHING as I am seeng (or not seeing!) now...

I precipitate with acetonitrile and ethanol... as I have seen no significant differences between the precipitation and delipidation protein amount, I am thinking of NOT precipitating proteins any more...

Thoughts?

:D

Thanks again to everyone helping out!
"Live Long and Prosper".

#6 mdfenko

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Posted 02 February 2010 - 08:51 AM

let us know how it works out.
talent does what it can
genius does what it must
i do what i get paid to do

#7 rachelhauser

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Posted 02 February 2010 - 09:53 AM

let us know how it works out.


Iīm buying the silver staining Kit from GE, should arrive in about 3 weeks (Brazil... everything takes forever to arrive, shipment and customs really suck!) and Iīll see how everythings goes, and Iīll post the results here. thanks again to everyone who helped out!
"Live Long and Prosper".

#8 mdfenko

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Posted 03 February 2010 - 08:28 AM

you could prepare your own silver stain solutions.

this post contains a downloadable formulation modified from merril's: silver stain formulation

Edited by mdfenko, 03 February 2010 - 08:29 AM.

talent does what it can
genius does what it must
i do what i get paid to do

#9 rachelhauser

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Posted 10 February 2010 - 07:50 AM

you could prepare your own silver stain solutions.

this post contains a downloadable formulation modified from merril's: silver stain formulation



Wow, thanks a lot for that protocol, people here in Brazil donīt seem to want to help other researchers, itīs a great surprise to find that it seems different in other countries. Iīll have to buy some of the reagents, but Iīll definitely give that protocol a shot, and let u know how it worked out! thanks again!!!
"Live Long and Prosper".




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