Hand-casting Gels for PAGE and SDS-PAGE Using mPAGE^® TurboMix Bis-Tris Gel Casting Kits

Section Overview

• Polyacrylamide Gel Electrophoresis (PAGE)
• Polyacrylamide Gel Chemistry
• Hand Casting Polyacrylamide Gels
• mPAGE^® TurboMix Bis-Tris Gel Casting Kit
• mPAGE^® TurboMix Quick Cast Protocol
• Preparing Your Samples and Running the Gel
• mPAGE^® TurboMix Bis-Tris Gels with MOPS & MES Running Buffer
• Ordering Information

Polyacrylamide gel electrophoresis (PAGE)

Polyacrylamide gel electrophoresis (PAGE) is a foundational technique used to separate proteins and other macromolecules by their electrophoretic mobility. Polyacrylamide gels are formed through the polymerization of acrylamide monomers and bis-acrylamide crosslinking molecules. Together, acrylamide and bis-acrylamide form pores through which proteins can migrate.

Polyacrylamide gels are composed of two parts: a stacking portion and a resolving portion. The stacking portion of the gel is a low percentage acrylamide that serves to concentrate samples into a single band before entering the resolving gel. The resolving gel is a higher percentage acrylamide that separates proteins by size. Acrylamide concentration is linearly related to pore size; a greater concentration of acrylamide will lead to smaller pores within the gel. Small pores are desirable for separating low molecular weight proteins.

Polyacrylamide gel chemistry

PAGE uses a discontinuous buffer system, wherein the gel buffer ion differs from the running buffer ion. The difference in electrophoretic mobility between these two ions forms a moving voltage gradient which proteins travel through. Tris-Glycine gel chemistry is the most commonly used PAGE system, which uses gels composed of Tris-HCl and running buffer composed of Tris base and glycine. Tris-Glycine gels operate in a highly alkaline environment which can lead to undesirable protein modifications like deamination and alkylation. As a result, protein bands may be distorted or lose resolution in Tris-Glycine gels.

In contrast, Bis-Tris gels use Bis-Tris and HCl in the gel buffer and MOPS or MES in the running buffer. Bis-Tris gels operate at a neutral pH which minimizes protein modification and promotes protein stability during the gel run. This leads to sharper protein band resolution and accuracy. Bis-Tris gels also have a longer shelf life than Tris-Glycine gels, which begin to hydrolyze over time. Bis-Tris gels have the flexibility to be combined with either MOPS- or MES-based running buffer; the difference in migration between these two ions results in different protein separation ranges. MES should be used when the protein of interest is small (<50 kDa) while MOPS should be used to resolve mid- to large-sized proteins.

It is also important to consider the type of sample buffer used during protein preparation. In Tris-Glycine gels, Laemmli buffer is typically used to denature and coat proteins in negatively charged SDS ions. Samples are then boiled at 100 °C to help facilitate denaturation. Heating Laemmli buffer to 100 °C causes the pH to become highly acidic and this combination of heat and acidity has been shown to cause protein cleavage preferentially at Asp-Pro peptide bonds (Rittenhouse and Marcus, 1984). This leads to apparent protein degradation products during electrophoresis. Conversely, Bis-Tris gels use an LDS sample buffer that maintains an alkaline pH during sample preparation and does not require heating above 70 °C to fully denature proteins. This preparation maintains protein integrity by minimizing Asp-Pro peptide bond cleavage.

Table 1. Comparison of Tris-Glycine and Bis-Tris gel chemistries.

	Tris-Glycine	Bis-Tris
Operating pH	9.5 (alkaline)	7.0 (neutral)
Sample buffer pH	5.2	8.5
Ion fronts	Tris (+) Glycine (-)	Tris (+) MOPS (-) MES (-)
Protein separation range	6 kDa – 400 kDa	6 kDa – 400 kDa
Protein stability during separation	Deamination and alkylation can occur	+++
Effect on Asp-Pro peptide bonds	Extended heating in SDS sample buffer causes cleavage	LDS sample buffer uses mild heating conditions and Asp-Pro bonds remain intact
Run time	Moderate	Fast
Shelf life	Limited	4 weeks +

Figure 1. Comparison of Tris-Glycine (left) and Bis-Tris (right) gels. Tris-Glycine and Bis-Tris gels were hand-cast with 12% acrylamide and allowed to polymerize overnight. The gels were loaded with identical E. coli lysate titrations (lanes 3-6), mPAGE^® unstained protein standard (lanes 2 and 7), and mPAGE^® color protein standard (lane 1). The gels were run in either Tris-Glycine or MOPS running buffer, stained with ReadyBlue™ protein gel stain for one hour, and destained with deionized water for one hour.

Hand casting polyacrylamide gels

While precast polyacrylamide gels can be purchased for specialized purposes, such as analyzing proteins of different sizes on an acrylamide gradient, many researchers choose to cast their own polyacrylamide gels by hand. Hand cast polyacrylamide gels are inexpensive compared to precast gels; however, preparation of reagents and casting equipment can be tedious and time consuming. Variation in gel buffer preparation can also lead to inconsistent gel performance and quality.

mPAGE^® TurboMix Bis-Tris Gel Casting Kit

The mPAGE^® TurboMix Bis-Tris Gel Casting Kit eliminates the variability and time-consuming nature of hand casting gels by supplying pre-mixed buffer and acrylamide solutions. The kit includes a 20% acrylamide Resolving Solution to form the resolving portion of an acrylamide gel. The Resolving Solution can be diluted with deionized water to the desired acrylamide percentage, from 8% - 15%. The kit also includes a 4% acrylamide Stacking Solution to form the stacking portion of an acrylamide gel. These solutions are formulated to allow for a Quick Casting method with no polymerization wait time between pouring the resolving and stacking gels. Below is a /US/endemo video and brief protocol.

Demo Video: Cast your own polyacrylamide gels using mPAGE^® TurboMix Bis-Tris Kits

mPAGE^® TurboMix QUICK CAST PROTOCOL

1. Prepare the resolving gel with the desired acrylamide percentage by pipetting mPAGE^® TurboMix Resolving Solution, deionized water, 10% ammonium persulfate (APS) and TEMED into a clean glass beaker or conical tube. Volumes in Tables 2-4 can be multiplied to cast several gels at once. NOTE: Add 10% APS and TEMED immediately before casting.

Table 2. Preparation of resolving solutions and stacking solutions for gel casting. Volumes listed are sufficient for casting one 7.4 x 8.2 cm mini-gel with 1 mm thickness and can be multiplied by n (desired number of gels) to cast multiple gels at once.

(For 1 mm thick mini-gel)
Resolving Gel		Stacking Gel
Gel percentage	8%	10%	12%	15%	4%
mPAGE® TurboMix Resolving Solution	2.4 mL	3.0 mL	3.6 mL	4.5 mL	n/a
mPAGE® TurboMix Stacking Solution	n/a	n/a	n/a	n/a	2 mL
De-ionized water	3.6 mL	3.0 mL	2.4 mL	1.5 mL	n/a
10% APS	30 µL	30 µL	30 µL	30 µL	20 µL
TEMED	3 µL	3 µL	3 µL	3 µL	2 µL

Table 3. Preparation of resolving solutions and stacking solutions for gel casting. Volumes listed are sufficient for casting one 7.4 x 8.2 cm mini-gel with 0.75 mm thickness and can be multiplied by n (desired number of gels) to cast multiple gels at once.

(For 0.75 mm thick mini-gel)
Resolving Gel		Stacking Gel
Gel percentage	8%	10%	12%	15%	4%
mPAGE® TurboMix Resolving Solution	1.8 mL	2.25 mL	2.7 mL	3.38 mL	n/a
mPAGE® TurboMix Stacking Solution	n/a	n/a	n/a	n/a	1.5 mL
De-ionized water	2.7 mL	2.25 mL	1.8 mL	1.12 mL	n/a
10% APS	22.5 µL	22.5 µL	22.5 µL	22.5 µL	15 µL
TEMED	2.25 µL	2.25 µL	2.25 µL	2.25 µL	1.5 µL

Table 4. Preparation of resolving solutions and stacking solutions for gel casting. Volumes listed are sufficient for casting one 7.4 x 8.2 cm mini-gel with 1.5 mm thickness and can be multiplied by n (desired number of gels) to cast multiple gels at once.

(For 1.5 mm thick mini-gel)
Resolving Gel		Stacking Gel
Gel percentage	8%	10%	12%	15%	4%
mPAGE® TurboMix Resolving Solution	3.6 mL	4.5 mL	5.4 mL	6.75 mL	n/a
mPAGE® TurboMix Stacking Solution	n/a	n/a	n/a	n/a	3.0 mL
De-ionized water	5.4 mL	4.5 mL	3.6 mL	2.25 mL	n/a
10% APS	45 µL	45 µL	45 µL	45 µL	30 µL
TEMED	4.5 µL	4.5 µL	4.5 µL	4.5 µL	2 µL

2. Prepare the stacking gel by pipetting mPAGE^® TurboMix Stacking Solution into a clean, separate glass beaker or conical tube and add required amount of TEMED and 10% APS. NOTE: Add 10% APS and TEMED immediately before casting.
3. Gently mix reagents, avoiding introduction of air bubbles into the gel mixture.
4. Using a serological pipette, fill each cassette to desired height with resolving gel.
5. Position serological pipette at the middle of the cassette and gently add the stacking gel, filling to the top of the short plate. A dip may occur where pipetting takes place but will level out.
6. Quickly and carefully insert the comb avoiding air bubble entrapment below the teeth.
7. Allow gels to polymerize for 1 hour.
8. Gels can be used immediately or wrapped in DI water-soaked paper towels and stored in an air-tight container at 4 °C for up to 4 weeks.

Preparing your samples and running the gel

Bis-Tris gels require LDS sample loading buffer, which is more alkaline than SDS sample buffer. Samples can be reduced using DTT or β-mercaptoethanol, or can be used non-reduced depending on your application. Prepare your samples according to Table 5.

Table 5. Example of PAGE sample preparation. Final concentration of LDS sample buffer should be 1X. Final concentration of DTT should be 0.1 M

	Reduced	Non-reduced
Sample	6.5 - X µL	7.5 - X µL
DI water	X µL	X µL
4X LDS buffer4X LDS buffer	2.5 µL	2.5 µL
1 M DTT	1 µL	N/A
Total volume	10 µL	10 µL

1. Heat samples for 10 minutes at 70 °C (do not boil).
2. Load samples and protein standard into wells.
3. Add an equal amount of 1X loading buffer into empty wells.
4. Gels can be run at 200 V until dye or protein standard reaches the end of the gel, approximately 30-60 minutes depending on gel percentage.

mPAGE^® TurboMix Bis-Tris Gels with MOPS and MES Running Buffer

mPAGE^® TurboMix Bis-Tris Gels are designed to work exclusively with MOPS or MES running buffer. Depending on which running buffer is used, very distinct separation patterns can be achieved. MOPS buffer can be used to fine-tune the separation of large and medium-sized proteins, whereas MES buffer provides optimal separation of smaller proteins. Refer to the migration charts (Figure 2) to determine which gel running buffer system is best suited for the intended separation range.

Figure 2. Migration charts for 8%, 10%, 12%, and 15% mPAGE^® TurboMix Bis-Tris gels with MOPS and MES SDS running buffer.