Sauer:bis-Tris SDS-PAGE, the very best
(→3.5X gel buffer)
|Line 69:||Line 69:|
Mix: 1/3.5 vol. of 3.5X bis-Tris gel buffer, acrylamide to 8% (30:2.0) or 12-15% (
Mix: 1/3.5 vol. of 3.5X bis-Tris gel buffer , acrylamide to 8% (30:2.0) or 12-15% (.), 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 25 μL of 10% APS per gel, mix in.
gel and .
Drain the , water , wick the water a .
Revision as of 10:42, 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.
Also available commercially.
200X running buffer reducing agent
1 M sodium bisulfite
add to 1X running buffer at 5 mM final concentration. If the leftover buffer sat for a few days, you can refresh it with more bisulfite before use.
This solution will contain meta-bisulfite. Depending on the vendor, your chemical powder may be labeled that way on the bottle to reflect its presence. It's is the right stuff.
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 changed 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 graduated conical tubes (e.g., pouring 15 mLs "by eye" to prepare 45 mLs of gel). Having the tank buffer and this gel buffer at pH 6.5 provides a more acidic environment and also more chloride ions than a pH 6.8 mixture, so try to get closer to 6.5, but don't sweat it.
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
Make sure you have enough 1X MES or MOPS tank buffer ready with relatively fresh sodium bisulfite added. When mixing from a cold 20X stock, the SDS is almost always out of solution, so mix the stock container well to distribute the SDS before pouring into the cylinder. When you add the water, it will re-dissolve.
Mix: 1/3.5 vol. of 3.5X bis-Tris gel buffer (or 1/3 of 1 M), acrylamide to 8% (30:2.0) or 12-15% (27.5:1), and water to final volume. I make 3.75 mLs for each Bio-Rad Protean gel, and use 3.5 mLs per gel. (if you made a 3X stock, adjust the buffer component volume). Unlike Tris-glycine gels, you can pre-mix this and keep it in the refrigerator for weeks.
Add 25 μL of 10% APS per gel, mix in.
Make sure your casting system and combs are ready...
Add 10 μL TEMED per gel and mix quickly.
Pour the gel to the appropriate level (using a 5 mL pipette works well, but a 1 mL pipetter may be more convenient), tip/tap the gel as you go to remove bubbles that get stuck, and cover with water from a squirt bottle to the top of the casting (being careful to get an even, undisturbed interface).
Let polymerize (will become evident as a new refractive layer forming right below the gel/water interface).
Drain the water, tip the gel past 90 degrees to collect remaining water to an upper edge of the casting, and wick the excess water off using a Kim-wipe. Don't worry about every small drop.
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.