Sharpen Up Your Protein Gels
Last week, Nick talked about about SDS-PAGE. Today I am going to tell you about tweak that will improve your SDS-PAGE protein gels.
It involves using Bis-Tris gel buffers. Although Bis-Tris adds a considerable cost to the technique, it has several advantages:
1. Bis-Tris gels are acidic, in contrast to the alkaline conditions found in conventional SDS-PAGE gels. This supresses cytosine reoxidation, which prevents proteins from cross-linking via di-sulphide bonds in the gel.
2. Sodium bissulphate, a reducing agent, is present throughout the buffer system. Unlike conventional PAGE, this means that the reducing environment is maintained all the way through the gel, which also helps to prevent disulphide bond formation.
3. Low molecular weight proteins do not run faster towards the end of the gel.
4. Conventional PAGE gels degrade after a month or two as the acrylamide breaks down to acryclic acid. This does not happen with Bis-Tris gels, which means they have a much longer shelf life.
Together, these factors mean that the resolution obtained on Bis-Tris SDS-PAGE gels is significantly greater than with conventional SDS-PAGE gels.
Here’s how to make a Bis-Tris gel:
5X low-MW running buffer Used for separating small proteins 2-50 kDa.
250 mM MES
250 mM Tris
5 mM EDTA
0.5% SDS
Sodium bisulfite to 5 mM (add fresh before run) from a 1M stock.
5X high-MW running buffer use for separating proteins >20 kDa.
250 mM MOPS
250 mM Tris
5 mM EDTA
0.5% SDS. add sodium bisulfite to 5 mM (add fresh before run) from a 1M stock.
200X running buffer reducing agent
1 M sodium bisulfite add to running buffer at 5 mM final concentration
3.5X gel buffer 1.25 M bis-Tris (pH 6.5-6.8 with HCl)
Note: bis-Tris is Bis(2-hydroxyethyl) aminotris (hydroxymethyl) methane (e.g. Sigma catalog# B 7535).
The method is not very different from the conventional methods of casting protein gels, just replace the tris buffer that you use in the stacking and resolving gels with the Bis tris buffer and omit SDS from the gel. Run at at a constant voltage of 150V.
Bis-tris gels were developed by Tim Updyke and Sheldon Engelhorn for Invitrogen. Similar gels are marketed by Invitrogen under the NuPAGE label.
If you try this, or already use them, let us know how these gels work for you.
Openwetware article on Bis-Tris gels
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Hi Bala
I was wondering if anyone else had tried to run this gel system on the smaller gel tanks, like the mini-Hoefer system (8×5.5cm). The gels run beautifully until about 1.5cm from the end, when things go all wavy, the dye front goes acidic, and basically the gel distorts (although after staining it returns back to normal). I’ve measured pH, run at low mA (as opposed to the patent which describes to run at 150V), and have remade the buffers very carefully. Only thing left to try is to replace the buffer halfway through the run. Any ideas ?
Liam, Infact i did face this problem – i was advised to run the gel at constant current (and i used a maximum voltage of 200V).The rationale being that the reservoir buffer completely displaces the gel buffer.The trick did work!
@Liam, I just had confused your problem with another one that i faced, at times the gel might run pretty slowly! which is because of the buffer exchange.Regarding your query,you could try adding some sodium bisulphite in the loading buffer (lamelli) – i did not have this problem when i used the NuPAGE loading buffer!
Bala
I’ll give it a try and report back.
Thanks
Liam
[...] all of our recent talk about how SDS-PAGE works and how to improve your gels, I wouldn’t want to give the impression that running protein gels is easy or [...]
When I tried this method I found that the protein bands are wavy. What could be the reason
I´m wondering which type of transfer buffer I should use for a bis-tris gel.
Would it be necessary to use the running buffer composition with the addition of methanol?
Do you have any hint?
The normal transfer buffer with methanol works well, however I’ll post an additional buffer composition that Invitrogen recommends, in a day or two.
I have the same problem with the mini-Hoefer system gels with the dye front getting wavy and acidic; I still have the problem using invitrogen’s LDS loading buffer. If you use a LOT of buffer in the bottom chamber, seems to be a bit better (and sometimes runs fine). I haven’t tried changing the buffer halfway. Anyone else have a solution to this problem?
Hi Bala,
I recently tried the BIS-TRIS system as described by u. I faced the same problem as described by others here. The dye front turned acidic and distorted at the end. I changed the whole running buffer and tried to run it again. But no luck.
Whether some one sorted out this problem? Kindly let me know.
Thanks in advance.
Guys, I’m still trying to get this system right! invitrogen suggests the following running buffer composition.Give it a try..
MOPS SDS buffer 20x (500ml)
MOPS 104.6g
Tris 60.6g
SDS 10.0g
EDTA 3.0g
Add water to makeup 500ml
I’ve tried making my own buffer or using commercial buffer; doesn’t make much if any difference. Rex – are you also using a Hoefer system?
Hi Bala,
Thanks for giving the buffer composition. Any other input from you will be greatly appreciated.
Dan,
I also use the Hoefer system. If u find out any remedy, let me know aswell. Did u try the buffer mentioned by Bala? How was it? Are you seeing sharper bands? My bands were not as good as what I see in Tris-Gycine system.
i’m pretty sure that’s the same buffer composition (just 20X, instead of 10X). My gels are great, nice sharp bands, etc, except for the problem at the bottom of the gel.
I have no problem with these gels. Run great at 180V constant voltage.
I am looking to try these gels out. I notice the invitrogen MOPS buffer recipe does not include the Sodium Bisulfite. Is this necessary?
@Foncy
If I’m right, the invitrogen MOPS buffer doesn’t have sodium bisulfite but they do recommend a special loading buffer which contains the reducing agent!