(1997) "Modification of the Laemmli Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis Procedure to Eliminate Artifacts on Reducing and Nonreducing Gels" Anal. We can also used a phosphate-based buffer if you need to cook your samples longer (it controls the pH to prevent acidic proteolysis) it's based on this reference: Cannon-Carlson, S., Tang, J. Using a lower temp, like 40-60 C to thaw frozen aliquots helps to prevent ruining the samples. A short heating of ~1 min at ~80-100 C is more than sufficient and only has to be done once to fully denature the proteins and coat them. Many people over-cook their proteins and cause brakdown, probably because Laemmli was solubilizing a protein pellet. You can use any of the the common Laemmli-like buffers. 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. 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). We have changed our recipe from the original to make it easier to make and use. Note: bis-Tris is Bis(2-hydroxyethyl) aminotris (hydroxymethyl) methane (e.g. Depending on the vendor, your chemical powder may be labeled that way on the bottle to reflect its presence. This solution will contain meta-bisulfite. If the leftover buffer sat for a few days, you can refresh it with more bisulfite before use. Oddly, this reagent can usually be found commercially as a pre-mixed solution that is cheaper than making it from scratch (the MES is expensive).Īdd to 1X running buffer at 5 mM final concentration. Use for separating small proteins 2-75 kDa. 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). Also, most deionized water sources have a low pH (from dissolved gas). If you are making your own stocks, use appropriate care when handling these toxins. 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. 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. 3.3 % cross-linker (found in 29:1 ratio stocks) will form smaller pores. 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. The amounts of bis-acrylamide typically varies in the stock depending on the size of proteins intended to be resolved and experience. In this unusual case, it is cheaper than buying the individual components and mixing them yourself.īuy or prepare a 30% acrylamide/bis-acrylamide stock. You can purchase pre-mixed MES and MOPS buffers from Boston Bioproducts. Note the broad separation range of the molecular weight markers. coli lysate with and without an induced protein. Sample bis-Tris MES-Tris Gels The first gel is of purified proteins.
Also, the same tank running buffer is used at both the cathode and anode. This allows gels to be cast and stored for a long time (diffusion doesn't ruin the stacking chemistry). The Stacking and Resolving layers of the gel use the same buffer. The result is higher resolution and a band distribution not unlike a gradient gel. 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. Additionally, a reducing agent, sodium bisulfite, is included in the running buffer and will migrate into the gel and maintain a reducing environment. In this protocol, in-gel cysteine reoxidation is suppressed by casting and running under slightly acidic (~pH 6.5) conditions favoring cysteine protonation.
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. 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. 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. The pH of the separating gel in “standard” SDS-PAGE (a.k.a.
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