Difference between revisions of "Sauer:bis-Tris SDS-PAGE, the very best"
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No need to pH.
No need to pH.
===<u>5X</u> high-MW running buffer===
===<u>5X</u> high-MW running buffer===
Revision as of 08:18, 23 December 2013
Submitted by Sean Moore
Based on work done by Tim Updyke and Sheldon Engelhorn for the Invitrogen Corporation (they bought Novex who developed it) and detailed in U.S. Patent 6,162,338. These gels are similar to those sold by Invitrogen as NuPAGE MES-Tris gels.
The pH of the separating gel in “standard” SDS-PAGE (a.k.a. Laemmli buffer system) is roughly 8-9 which is conducive to the deamination and alkylation of proteins, as well as reoxidation of reduced cysteines during electrophoresis. What this means is that your protein will form disulfide crosslinks during the stacking event because the protein migrates into the gel away from the reducing reagent in the sample buffer, and gets focused to a high concentration. Acrylamide gels cast in alkaline buffers are also unstable during long term storage, breaking down to acrylic acid after 1 to 2 months resulting in loss of pore size, poor resolution, and modified proteins.
In this protocol, in-gel cysteine reoxidation is suppressed by casting and running under slightly acidic (~pH 6.5) conditions favoring cysteine protonation. Additionally, a reducing agent, sodium bisulfite, is included in the running buffer and will migrate into the gel and maintain a reducing environment. Another feature of this gel system is that the lower MW proteins near the buffer front do not accelerate towards the end of the run to the same degree as in Laemmli buffers. The result is higher resolution and a band distribution not unlike a gradient gel.
The Stacking and Resolving layers of the gel use the same buffer. This allows gels to be cast and stored for a long time (diffusion doesn't ruin the stacking chemistry). Also, the same tank running buffer is used at both the cathode and anode.
Sample bis-Tris MES-Tris Gels The first gel is of purified proteins. The second gel is of total E. coli lysate with and without an induced protein. Note the broad separation range of the molecular weight markers.
You can purchase pre-mixed MES and MOPS buffers from Boston Bioproducts. In this unusual case, it is cheaper than buying the individual components and mixing them yourself.
Buy or prepare a 30% acrylamide/bis-acrylamide stock. The amounts of bis-acrylamide typically varies in the stock depending on the size of proteins intended to be resolved and experience. Having the bis-acrylamide crosslinker at a lower concentration (e.g., 2.6 % found in 37.5:1 ratio mixtures) will allow larger pores to form and resolve larger proteins better. 3.3 % cross-linker (found in 29:1 ratio stocks) will form smaller pores. In practice, it's easier to use a 37.5:1 stock and change the final concentration (from ~12.5-15%) than it is to maintain several stocks. Alternatively, you can have a separate stock of dissolved bis-acrylamide and spike it into the final gel mixture to vary the cross-linker level.
If you are making your own stocks, use appropriate care when handling these toxins. Also, most deionized water sources have a low pH (from dissolved gas). This is good for acrylamide stocks and helps to prolong their shelf life, so don't adjust the pH with base (it isn't buffered and you'll cook your acrylamide).
5X low-MW running buffer
Use for separating small proteins 2-50 kDa.
250 mM MES 250 mM Tris 5 mM EDTA 0.5% SDS No need to pH.
Oddly, this reagent can be found commercially as a pre-mixed solution that is cheaper than making it from scratch (the MES is expensive).
5X high-MW running buffer
use for separating proteins >20 kDa.
250 mM MOPS 250 mM Tris 5 mM EDTA 0.5% SDS No Need to pH.
add sodium bisulfite to 5 mM (add fresh before run) from a 1M stock. It's stinky.
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).
We have changes our recipe slightly to make it easier to make and use. Prepare 1.0 M bis-Tris as a "3X". Using thirds helps when making the gel solutions in graduate conical tubes.
I routinely use the "standard" Laemmli 3X buffer. I think it does a better job reducing samples because of the higher pH.
Casting and running gels
Mix: 1/3.5 vol. of 3.5X bis-Tris gel buffer, acrylamide to 8% (30:2.0) or 12-15% (30:0.8), and water to final volume. I make 3.75 mLs for each Bio-Rad Protean gel, and use 3.5 mLs per gel.
Add 25 μL of 10% APS per gel, mix in.
Add 7 μL TEMED
Pour gel and cover with ~1 mL of bis-Tris buffer saturated butanol.
Drain the butanol, rinse with water, wick the water from between the plates with a shred of filter paper.
1X bis-Tris gel buffer, acrylamide solution to 4%, water.
Optional: Bromophenol Blue solution to make the stacking gel blue. It really helps when loading samples and doesn't affect the performance.
I make 2.5 mLs stacking per gel which is a bit too much.
Don't add APS until you are ready to start the polymerization. At the lower pH, the APS will start to polymerize the stacking gel while your resolving gel sets and will be all goopy.
Add 15-20 μL 10% APS, mix.
Add 7-10 μL TEMED, mix, pour onto the resolving layer, insert comb.
Rinse with water to remove unpolymerized acrylamaide. Remove comb.
Fill both upper and lower buffer chambers with either MES-Tris or MOPS-Tris buffer.
Run at 150V constant. If you're running two or more gels in the same tank, the temperature can get quite high. In these cases I run with constant current, you will see the voltage rise quite a bit a the electrophoresis progesses. The Bromophenol blue runs around 3-5 kDa with the MES-Tris buffer system.