Gel electrophoresis tips and troubleshooting
Gel electrophoresis tips and troubleshooting
Laemmli separating gel buffer - Buffer chloride concentration (not pH) greatly affects separation. High chloride improves stacking, but smaller proteins run with the front. Low chloride facilitates separation of small proteins at the expense of
stacking. Adding HCl volumetrically from reagent con HCl is simpler and more reliable than adjusting with a pH meter. (see recipes)
Sample buffer - Sample (cracking) buffer has little affect on separation. pH 6.8 Tris buffer is very poor for reducing proteins (the pH is about 4.5 at 100oC!). Choose a buffer with a higher pH at 100oC. more sample tips
Casting stacking gel - Pour the stacking gel solution, then warm it with a hair dryer. This gives uniform and "complete" polymerization.
Mild cleaning solution - 1% SDS + 1% oxalic acid (overnight soak) cleans scale, rust, fingerprints and traces of grease from gel plates. Wash plates thoroughly before soaking. SDS gradually hydrolyzes; the solution becomes turbid in about six months.
Gel thickness - Minimizing gel thickness facilitates staining. With silver development, staining time increases with roughly the square of gel thickness. The thinnest available teflon sheet is 0.75mm. Thinner gels would be hard to handle. On the other hand
0.5mm or 0.5mm teflon sheet might improve silver staining substantially!
Advantages of silver staining - Silver development ("staining") is more sensitive than Coomassie Blue or ethidium bromide. It is quick and cheap (primary cost, as with Coomassie Blue, is organic solvents). Staining is "permanent"; cheap flatbed scanners record data. Commercial staining kits are superfluous. Silver detects more components giving a clearer idea of sample composition (of course one may be happier not knowing).
Protein thiols - One normally reduces proteins before SDS gel electrophoresis. Proteins with several cys residues can reoxidize during electrophoresis creating "smears". Large thiol rich proteins (e.g. serum albumin and rubisco L subunit) are especially prone to reoxidation. Reducing with DTT (see advice) and alkylating with iodoacetamide or N-ethylmaleimide prevents this.
Stability of polyacrylamide - I recently ran a Laemmli gel after it sat on the bench at room temperature for about a month. The gel appears to have partially hydrolyzed. It ran slowly (electroendosmosis). I stopped it when the dye front was ~3/4 of the way down. The gel had swollen appreciably, particularly below the "front" (higher pH region). Despite the apparent presence of carboxyl groups, the gel stained satisfactorily with silver though the surface was messy and the gel was brittle.
Polyacrylamide vs. agarose - Polyacrylamide resolves better (smaller pore size) and gives less background staining. Pore size limitations prevent polyacrylamide separations of larger macromolecules. Agarose is more convenient. Literature exaggerates it's purity.
Reproducible electrophoresis - Geometry and buffer composition determine electrical resistance of electrophoretic systems. Assuming constant geometry, at a given voltage current should be the same from gel to gel (a handy quality control parameter). Buffer variation leads to current irreproducibility. See weigh and pour recipes
Fluorescence detection - Have you seen the Dark Reader? I hoped that media such as polycarbonate centrifuge tubes and agarose would fluoresce less (reduced background fluorescence) with blue illumination - no such luck. Molecular Probes makes many useful dyes including Sypro ruby for proteins and Sybr Gold for staining nucleic acids in gels.
Gel problems
No SDS in wells - protein precipitates as it enters running gel
High salt concentration in some samples - "wide lanes" and localized distortion
Nothing on gel - failed to load or ran the wrong way (be sure to follow tracking dye as it begins to migrate).
Gel swells abnormally and is brittle - aging (polyacrylamide has partially hydrolyzed).
Gel is gooey and sticks to glass - you forgot crosslinker
Silver development slow - add more formaldehyde to developer or use fresh formaldehyde (problem may be aldol condensation, methanol is added to stabilize formaldehyde solutions, it's volatile and gradually vaporizes. Formaldehyde also oxidizes and is light sensitive.)
e-mail: llane1@unl.edu