The pH and ionic composition of the buffer determine power requirements and heavily influence the separation characteristics of a polyacrylamide gel. This section provides guidelines for choosing the appropriate buffers for different gel types for electrophoresis.
- Cast the gel
- Prepare the sample (sample buffer)
- Fill the electrode reservoirs (running buffer)
Most common PAGE applications utilize discontinuous buffer systems (see Niepmann 2007 for a review of discontinuous buffer systems), where two ions differing in electrophoretic mobility form a moving boundary when voltage is applied (see Protein Electrophoresis Equipment). Proteins have an intermediate mobility, so they stack, or concentrate, into a narrow zone at the beginning of electrophoresis. As that zone moves through the gel, the sieving effect of the gel matrix causes proteins of different molecular weights to move at different rates. Varying the types of ions used in the buffers changes the separation characteristics and stability of the gel. The table below summarizes the various types of gel and buffer systems available.
|Gel Type||Selection Criteria||Buffers||Precast (Format) Gels||Handcast|
|Tris-HCI, pH 8.6||Easy to prepare, reagents inexpensive and readily available; best choice when switching between precast and handcast gels and need to compare results||Laemmli||Tris/glycine/SDS||•||•||•|
|TGX™||Laemmli-like extended shelf life gels; best choice when long shelf life is needed and traditional Laemmli separation patterns are desired||Laemmli||Tris/glycine/SDS||•||•||—|
|Tris-HCl Stain-Free™||Laemmli-like gels with trihalo compounds for rapid fluorescence detection without
|TGX Stain-Free™||Laemmli-like extended shelf life gels with trihalo compounds for rapid fluorescence
detection without staining
|Bis-Tris, pH 6.4||Offer long shelf life, but require specialized buffers which may be costly||XT||XT MOPS or XT MES||—||•||—|
|Tris-acetate, pH 7.0||Offer best resolution of high molecular weight proteins; useful in peptide sequencing or mass spectrometry applications||XT or Tricine||XT Tricine or Tris/Tricine/SDS||•||•||•|
|Tris-HCI, pH 8.6||Retention of native protein structure, resolution of proteins with similar molecular weight||Native||Tris/glycine||•||•||•|
|TGX||Laemmli-like extended shelf life gels; best choice when long shelf life is needed and traditional Laemmli separation patterns are desired||Native||Tris/glycine||•||•||—|
|Tris-HCl Stain-Free||Laemmli-like gels with trihalo compounds for rapid fluorescence detection without staining||Native||Tris/glycine||—||•||—|
|TGX Stain-Free||Laemmli-like extended shelf life gels with trihalo compounds for rapid fluorescence detection without staining||Native||Tris/glycine||—||•||—|
|Tris-acetate, pH 7.0||Offer best separation of high molecular weight proteins and protein complexes||Native||Tris/glycine||—||•||•|
|Tris-Tricine||Optimized for separating peptide and proteins with molecular weight <1,000||Tricine||Tris/Tricine/SDS||•||•||•|
|IEF||Cast with Bio-Lyte ampholytes, allow separation by protein pl; contain no denaturing agents, so IEF is performed under native conditions||50% glycerol||IEF cathode and IEF anode buffers||•||•||—|
|Zymogram||Cast with gelatin or casein, which acts as a substrate for proteases that are separated in the gel under nondenaturing conditions||Zymogram||Tris/glycine/SDS||•||•||•|
The Laemmli system has been a standard system for SDS and native PAGE applications for years. Many researchers use Tris-HCl gels because the reagents are inexpensive and readily available; Precast gels are also readily available in a wide variety of gel percentages.
This discontinuous buffer system relies on the stacking effect of a moving boundary formed between the leading ion (chloride) and the trailing ion (glycinate). Tris buffer is the common cation. Tris-HCl gels can be used in either denaturing SDS-PAGE mode (using Laemmli sample buffer and Tris/glycine/SDS running buffer) or in native PAGE mode (using native sample and running buffers without denaturants or SDS).
Tris-HCl resolving gels are prepared at pH 8.6–8.8. At this basic pH, polyacrylamide slowly hydrolyzes to polyacrylic acid, which can compromise separation. For this reason, Tris-HCl gels have a relatively short shelf life. In addition, the gel pH can rise to pH 9.5 during a run, which can cause proteins to undergo deamination and alkylation; this may diminish resolution and complicate post-electrophoresis analysis.
Bio-Rad has developed modified Laemmli gels with a proprietary modification that extend shelf life to 12 months and allow gels to be run at higher voltages without producing excess heat. The formulation of the TGX (Tris-Glycine eXtended shelf life) precast gels yields run times as short as 15 min and Laemmli-like separation patterns with exceptionally straight lanes and sharp bands. TGX gels offer excellent staining quality and transfer efficiency (with transfer times as short as 15 min), and they do not require special, expensive buffers. Like Tris-HCl gels, TGX gels use a discontinuous buffer system, with glycinate as the trailing ion, and so are compatible with conventional Laemmli and Tris/glycine/SDS buffers. These are the best choice when long shelf life is needed and traditional Laemmli separation patterns are desired.
Bio-Rad's TGX Stain-Free gels are Laemmli-like, extended shelf life gels that allow rapid fluorescent detection of proteins with the Gel Doc EZ imaging system, eliminating staining and destaining steps for results in 25 min (see Protein Staining for more details about the stain-free technology).
These systems employ chloride as the leading ion and MES or MOPS as the trailing ion. The common cation is formed from Bis-Tris buffer. The gels are prepared at pH 6.4, which enhances gel stability. Running the same Bis-Tris gels with either MES or MOPS denaturing running buffer produces different migration patterns: MES buffer is used for small proteins, and MOPS buffer is used for mid-sized proteins.
Precast Bis-Tris gels (for example, Criterion XT Bis-Tris gels) offer extended shelf life (compared to Tris-HCl gels) and room temperature storage. These gels are popular because of their stability; they require special buffers, however, and gel patterns cannot be compared to those of Tris-HCl gels.
Common reducing agents such as βME and DTT are not ionized at the relatively low pH of Bis-Tris gels and so do not enter the gel and migrate with the proteins. Alternative reducing agents are, therefore, used with Bis-Tris gels to maintain a reducing environment and prevent protein reoxidation during electrophoresis.
This discontinuous buffer system uses acetate as the leading ion and Tricine as the trailing ion and is ideally suited for SDS-PAGE of large proteins. Tris-acetate gels can be used for both SDS- and native PAGE and, like Bis-Tris gels, offer extended shelf life and room temperature storage. Because of their lower pH, these gels offer better stability than Tris-HCl gels and so are best suited for peptide sequencing and mass spectrometry applications.
One of the drawbacks to using SDS in a separation system is that excess SDS runs as a large front at the low molecular weight end of the separation. Smaller polypeptides do not separate from this front and so do not resolve into discrete bands. Replacing the glycine in the Laemmli running buffer with Tricine yields a system that separates the small SDS-polypeptides from the broad band of SDS micelles that forms behind the leading-ion front. Proteins as small as 1–5 kD can be separated in Tris-tricine gels.
Isoelectric focusing (IEF) separates proteins by their net charge rather than molecular weight. IEF gels are cast with ampholytes, amphoteric molecules that generate a pH gradient across the gels. Proteins migrate to their isoelectric point (pI), the pH at which the protein has no net charge. IEF gels contain no denaturing agents, so IEF is performed under native conditions.
Zymogram gels contain either gelatin or casein, which are substrates for various proteases. Samples are run under denaturing, but non-reducing conditions, then allowed to renature and consume the substrate. Samples with proteolytic activity can be visualized as clear bands against a blue background after using Coomassie Brilliant Blue R-250 stain.
This tutorial shows how to use Bio-Rad's Mini-PROTEAN TGX precast gels with the Mini-PROTEAN Tetra cell. Mini-PROTEAN TGX precast gels are long shelf life precast gels that use standard Tris/Glycine buffers.