Author Topic: Coomassie Blue staining - no bands  (Read 17720 times)

rachelhauser

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Coomassie Blue staining - no bands
« on: November 14, 2011, 10:58:37 AM »
Hi again everyone, once again I´m in need of ANY suggestions/ideas/criticisms...

I don´t know if anyone remembers, but I was having problems with my samples, they were too dilute, and silver staining is a pain... so I solved my "too dilute sample" problem by using Vivaspin concentrators to remove salts and concentrate my sample (fish bile) in a single step.

Thus, I happily set about running my 1D 12.5% mini gels (Mini VE system by GE Healthcare) for subsequent trypsinization/LC-MS/MS identification of my fish bile proteins.

HOWEVER (you knew it was coming...)...

I set about testing my clean-up procedures. As it was a test, I just put 10 uL of each sample on a gel (treated in different ways) and ran them, just to see what would happen. The attached picture (just a cel pic) shows 8 lanes, all differently treated before electrophoresis, all the same VOLUME applied (10 uL), but not the same amount of protein (since I didn´t quantify, I just wanted to see what my clean-up procedures would do to my samples).

After running the gels, however, I could only get this profile (VERY faint bands at first) after 2 days staining in Coomassie Blue (8% ammonium sulfate, 0.8% phosphoric acid, 0.08% Coomassie Blue G-250, 20% methanol, 500 ml). After another 2 days kept in a ziploc bag, I can see a very good 1D profile. So, things turned out OK, but only after two days staining, one afternoon destaining and 2 days in a ziploc bag in 2% acetic acid. (Oh, and I fix the proteins before staining in 40% ethanol/10% acetic acid for about 30 min).

I thought that the 2 days between staining and getting a good profile was strange, but went ahead and applied 30 ug of a few samples (after quantifying by 2D quant-Kit AND regular Lowry methods, both were almost the same, and very consistent, giving me up to 10 ug/uL, perfect for applying small amounts on mini gels!) on two other gels.

I used the same Coomassie recipe, and am now destaining them, and will secure them in ziplocs to see what happens, but it´s been about 2 days and no bands yet, only a few VERY faint ones, I have to look very hard to find them.

So, after this weird experience, today I tested another coomassie recipe, 0.1% Coomassie blue, 10% acetic acid, 40% ethanol on another gel, which at the end was much "bluer" than the original stain I used, it´s a bit more concentrated obviously. So I stained a gel, and the whole gel turned REALLY BLUE, with no discernible bands, just a blue gel. I´ve been destaining for most of the afternoon, and can see the standards OK, and again, some VERY, VERY FAINT bands... (I also fixed the gel prior, just in case...)

Any ideas? Does it usually take a few days to see something? I used to see bands right away. Now, I know staining gets more and more intense for at least 7 days after the destaining and after I put the gels in a ziploc bag or maintain them in 2% acetic acid, but, frankly, this is just weird... As I fixed the protein before, I could, in theory, destain and stain them a few times in other coomassie stains, right??
 
Thank in advance for any insights...!  ;D

P.s. - In the attached pic, what I´m applying now are samples treated as in lane 8, Sonicated + centrifuged + delipidized + Vivaspin sample, which looks fine there.

p.s. 2 - I don´t think it´s a quantification problem, I ran a rat brain gel with 20 ug total protein quantified in the same way (both 2D quant kit and Lowry method) and got a very good 1D profile the same day I stained, with big fat bands, not the faint bands I´m seeing now for fish bile.


« Last Edit: November 14, 2011, 11:18:00 AM by rachelhauser »

dfried

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Re: Coomassie Blue staining - no bands
« Reply #1 on: November 14, 2011, 09:46:42 PM »
The fact that you can get the brain samples to stain normally at least tells you that the stain is working, but it sounds like it is not very sensitive.  It could be something in your gels that's interfering; do you cast them yourself?  Coommassie shouldn't be this hard.  It sounds like you're making it up from the powder.  Can you try some premade stuff? You might find better advice in the electrophoresis forum, too.

David

rachelhauser

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Re: Coomassie Blue staining - no bands
« Reply #2 on: November 15, 2011, 02:11:59 AM »
Hello David, yes, I cast the gels myself (recipes from GE 2D Handbook), all new solutions and reagents, electrophoresis grade. I make the coomassie from the powder, yesterday I opened a new one, sealed and everything, just in case... I can't get my hands on the pre made stuff unfortunately, no one uses that here, it's waaaaaay too expensive... everyone makes up their own coomassie solutions.

Do you think I should destain and stain again with some other recipe? The proteins are fixed, so they should be fine, right?

Thank you!  :D

p.s. - Did I submit to the Western Blotting forum? It says here I'm on the 2D electrophoresis one, is this a bug or something?

 :)



Sjouke Hoving

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Re: Coomassie Blue staining - no bands
« Reply #3 on: November 15, 2011, 03:28:57 AM »
Here is a protocol for colloidal Coomassie staining: (cheap and sensitive)
1. Fixing 50% (v/v) ethanol, 3% (v/v) phosphoric acid 3 hours
2. Washing Distilled water 3 x 30 minutes
3. Staining – first step 34% (v/v) methanol, 3% (v/v) phosphoric acid, 17% (w/v) ammonium sulfate 1 hour
4. Staining – second step2 Coomassie Blue G-250 (350 mg/L into solution 3) 1 – 5 days (after a few hours bands become already visible, but at least O7N staining for best result)
5. Washing Distilled water 3 x 30 minutes

sjouke

rachelhauser

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Re: Coomassie Blue staining - no bands
« Reply #4 on: November 15, 2011, 04:09:26 AM »
Hello Sjouke, I´ll try your recipe, do you think I can try with the same gels I stained and destained yesterday?

Thank you!

Sjouke Hoving

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Re: Coomassie Blue staining - no bands
« Reply #5 on: November 15, 2011, 05:15:10 AM »
Yes sure, you can easily re-stain gels with this protocol.

sjouke

rachelhauser

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Re: Coomassie Blue staining - no bands
« Reply #6 on: November 15, 2011, 09:09:12 AM »
Hello Sjouke, I just left the gels (the 2 from yesterday and the 2 from Sunday) in the first step of the staining solution from your recipe... in theory I could destain and stain indefinitely, right, since the proteins are well fixed in the gels?

If your stain doesn't work I'm thinking of either trying a new recipe, that uses the microwave for a few minutes in a few steps, to see if anything turns up, or simply shooting myself in the head since I have to have final results in February...  :-\




Sjouke Hoving

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Re: Coomassie Blue staining - no bands
« Reply #7 on: November 16, 2011, 01:03:50 AM »
Yes, the proteins are well fixed... you could re-stain with silver as alternative...

Your last option is no option, it is not worth it ;)

rachelhauser

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Re: Coomassie Blue staining - no bands
« Reply #8 on: November 17, 2011, 04:05:47 AM »
Sjouke, with all due respect, I LOVE YOU! Thank you so much, the coomassie blue staining recipe you gave me worked perfectly, I can see all my protein bands with just the overnight staining! You saved my life (and my PhD title too!)

 :D

I have just a tiny question, the staining solution is now transparent, not blue at all, and it´s only day one of staining, should I add more coomassie blue, or is this normal? I´d like to leave it for a few more days in order to stain the bands a bit more.

Thank you SO much!!!



rachelhauser

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Re: Coomassie Blue staining - no bands
« Reply #9 on: December 06, 2011, 11:12:10 AM »
Just wanted to let everyone know, this is an EXCELLENT coomassie staining method, I did 2 exact same gels and compared the coomassie staining, with my old protocol and the new one, and WOW, the one Sjouke gave me is MUCH better!

I have a question though, would it be ok to leave the gel overnight in the fixing solution (ethanol + phosphoric acid?), or does it have to be just the 3 hours?

Thank you!

Sjouke Hoving

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Re: Coomassie Blue staining - no bands
« Reply #10 on: December 07, 2011, 12:17:03 AM »
You're welcome...

Of course, it is no problem to leave the gel in the fixing solution O/N. I have even left it over the weekend in fixing solution, depending on the work schedule.


sjouke

rachelhauser

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Re: Coomassie Blue staining - no bands
« Reply #11 on: December 15, 2011, 12:02:11 PM »
Sjouke, I have another small question, can I reuse any of the solutions (ethanol + phosphoric acid, methanol + phosphoric acid + ammonium sulphate and the latter + coomassie?)

I found that I have no more ammonium sulphate and as it´s nearing the end of the year I don´t know when the order I placed of that will arrive, so I was just wondering. The same with my ethanol, I´m now using a non-molecular biology grade to fix the proteins... hope that doesn´t interfere too much.

Thank you!
Rachel

Sjouke Hoving

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Re: Coomassie Blue staining - no bands
« Reply #12 on: December 16, 2011, 01:33:07 AM »
In an emergenc case I guess you can re-use the solutions, but it is not really recommended. The methanol might slowly evaporate from the colloidal solution, which changes than the propert of the staining solution. Fixing solution take up a lot of contaminants from the gels (buffer components, SDS) and should also not be re-used in general.
All solutions are rather cheap, so we always used to make fresh solutions.

sjouke

rachelhauser

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Re: Coomassie Blue staining - no bands
« Reply #13 on: December 19, 2011, 10:41:52 AM »
Hi Sjouke, thanks for your input, I just got hold of some reagents so I´m good until January at least, it´s interesting to know the WHY of things, so I really appreciate your answer with explanations! :)