16 replies to this topic

[Fredriksen]

I have been running about a million gels with tons of different conditions to try to visualize a set of proteins in the 1-6 kD range. They are membrane proteins that I've been trying to express in E. Coli, and they are rather hydrophobic. I've been using the Tricine gel method described by Schagger in Nature Protocols, (2006) vol 1 p16-22. So far, I have not been able to see any bands whatsoever below 10 kD. I've tried the following:

10% Acrylamide:BisAcryl 37.5:1
15% Acrylamide:BisA 19:1
15% Acrylamide:BisA 19:1 with 8M Urea and 10% spacer gel w/37.5:1 A:BA
The stacking gel is 4%

Using different concentrations of cells.
Using different concentrations of SDS in sample buffer (otherwise I used sample buffer recipe exactly as in the paper mentioned above)
Boiling samples for 5 min
Heating samples to 65 deg for 20 min
incubating samples for 1 hour at 37 deg
Visualizing with Coomassie
Visualizing with silver stain

I am using mini-gels that are about 13x8cm that I've been casting myself. I start the run at 30 V and then when the ladder is entirely in the separating gel, I step the voltage up to 200 V

I am going to try tomorrow to run the samples with 8M Urea in the sample buffer. After that I will try bis-Tris gels (ordered from BioRad). If none of this is going to work, please tell me. I can see the 4kD and 1kD bands on my protein ladder resolved perfectly fine, I just can't see anything below 10kD on my samples.

[genehunter]

Is there any possibility that your proteins turned aggregated after cooking with denaturing mixture with SDS?

[mdfenko]

have you tried the original method of shagger and von jagow:

Schagger, H. and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368-379.

we used this method and were able to resolve sub 1kDa peptides as well as higher.

for the lower mw peptides we used 16.5% running gel, 10% spacer and 4% (i think, someone borrowed my methods book so i can't check right now) stack.

[Fredriksen]

mdfenko, on Jun 16 2009, 09:42 AM, said:

have you tried the original method of shagger and von jagow:

Schagger, H. and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368-379.

we used this method and were able to resolve sub 1kDa peptides as well as higher.

for the lower mw peptides we used 16.5% running gel, 10% spacer and 4% (i think, someone borrowed my methods book so i can't check right now) stack.

Thanks, I will look at this paper closely and see what I can change in my procedure today. One thing I am noticing is that the gels are larger in these papers than what I use (Bio-Rad mini-gels, about 8x8cm, d'oh I accidentally put the wrong size in my opening post). Did you use large gels, or did you use mini-gels?

Edited by Fredriksen, 16 June 2009 - 09:46 AM.

[Fredriksen]

genehunter, on Jun 16 2009, 08:28 AM, said:

Is there any possibility that your proteins turned aggregated after cooking with denaturing mixture with SDS?

I'm not sure, which is why I tried incubation at different temperatures. Do you have any suggestions for avoiding this? These are whole cells I'm trying to check, so I am weary of not giving any sort of heat treatment.

[mdfenko]

Fredriksen, on Jun 16 2009, 01:43 PM, said:

Thanks, I will look at this paper closely and see what I can change in my procedure today. One thing I am noticing is that the gels are larger in these papers than what I use (Bio-Rad mini-gels, about 8x8cm, d'oh I accidentally put the wrong size in my opening post). Did you use large gels, or did you use mini-gels?

we used in both mini and "large" gels (i put it in quotes because your large is our small gel, our large gel is affectionately referred to as a blanket). we adjust the volumes so that we get approximately the same ratio of run/spacer/stack, give or take a little.

boiling for 3-5 min or heating at 65C for 10-20 minutes should be sufficient.

some samples did not show up with a single silver stain. we had to double stain to make the bands appear.

[mastermi]

Fredriksen, on Jun 16 2009, 07:43 PM, said:

mdfenko, on Jun 16 2009, 09:42 AM, said:

have you tried the original method of shagger and von jagow:

Schagger, H. and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368-379.

we used this method and were able to resolve sub 1kDa peptides as well as higher.

for the lower mw peptides we used 16.5% running gel, 10% spacer and 4% (i think, someone borrowed my methods book so i can't check right now) stack.

Thanks, I will look at this paper closely and see what I can change in my procedure today. One thing I am noticing is that the gels are larger in these papers than what I use (Bio-Rad mini-gels, about 8x8cm, d'oh I accidentally put the wrong size in my opening post). Did you use large gels, or did you use mini-gels?

For Schägger-gels I use larger glass plates only (they are selfmade and about 12x22 cm in size). So the running distance is about 2.5 times longer than with mini-gels...

[Fredriksen]

mdfenko, on Jun 16 2009, 01:01 PM, said:

Fredriksen, on Jun 16 2009, 01:43 PM, said:

Thanks, I will look at this paper closely and see what I can change in my procedure today. One thing I am noticing is that the gels are larger in these papers than what I use (Bio-Rad mini-gels, about 8x8cm, d'oh I accidentally put the wrong size in my opening post). Did you use large gels, or did you use mini-gels?

we used in both mini and "large" gels (i put it in quotes because your large is our small gel, our large gel is affectionately referred to as a blanket). we adjust the volumes so that we get approximately the same ratio of run/spacer/stack, give or take a little.

boiling for 3-5 min or heating at 65C for 10-20 minutes should be sufficient.

some samples did not show up with a single silver stain. we had to double stain to make the bands appear.

Did you double stain with Coommassie/Silver, or did you just silver stain for a long time?

[mdfenko]

Fredriksen, on Jun 16 2009, 08:21 PM, said:

Did you double stain with Coommassie/Silver, or did you just silver stain for a long time?

we stain with silver twice (no need to destain in between but can be done). the background may end up being dark but the method we use still maintains translucence.

[Fredriksen]

mdfenko, on Jun 17 2009, 07:47 AM, said:

Fredriksen, on Jun 16 2009, 08:21 PM, said:

Did you double stain with Coommassie/Silver, or did you just silver stain for a long time?

we stain with silver twice (no need to destain in between but can be done). the background may end up being dark but the method we use still maintains translucence.

I suppose a longer development time just doesn't cut it? I'm using a silver stain kit from GE.

[mdfenko]

Fredriksen, on Jun 17 2009, 03:39 PM, said:

I suppose a longer development time just doesn't cut it? I'm using a silver stain kit from GE.

no. longer development time just makes the background darker. restaining appears to enhance silver binding.

we use a homemade (labmade?) modification of merril's method (if you want, i'll give you the reference when i get my book back, i was supposed to have it yesterday).

[Fredriksen]

mdfenko, on Jun 17 2009, 01:00 PM, said:

Fredriksen, on Jun 17 2009, 03:39 PM, said:

I suppose a longer development time just doesn't cut it? I'm using a silver stain kit from GE.

no. longer development time just makes the background darker. restaining appears to enhance silver binding.

we use a homemade (labmade?) modification of merril's method (if you want, i'll give you the reference when i get my book back, i was supposed to have it yesterday).

That would be awesome if you could do that for me

[mdfenko]

as soon as i get my book back, then, i'll give you the reference and our formulation with modifications.

[Fredriksen]

mdfenko, on Jun 18 2009, 09:39 AM, said:

as soon as i get my book back, then, i'll give you the reference and our formulation with modifications.

Had you ever used any of the SYPRO staining methods for the small peptides as well? I tried double silver staining last week and I still could not see below 10kD. I guess I either need your formulation or I need to try something else.

Edited by Fredriksen, 02 July 2009 - 06:56 PM.

[mdfenko]

i'm sorry that i haven't posted yet. i am still waiting for the return of my methods book (and getting really p.o.ed that my requests have been ignored). i hope to be able to post soon.