Hand Casting Polyacrylamide Gels

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Overview

Handcast gels must be prepared from acrylamide and bisacrylamide monomer solutions; the component solutions are prepared, mixed together, and then poured between two glass plates to polymerize. This section provides general tips for handcasting gels, protocols for handcasting single and gradient gels, and a list of Bio-Rad products available for handcasting gels.

 

Related Topics: Polyacrylamide Gels, Buffer Systems and Gel Chemistries, and Protein Standards.

 

Find more information on hand casting single and gradient SDS-PAGE gels in the Protocols section below.

General Tips for Handcasting

  • Acrylamide and bisacrylamide are neurotoxins when in solution. Avoid direct contact with the solutions and clean up spills.
  • For casting multiple gels, use the Mini-PROTEAN® 3 multi-casting chamber, PROTEAN® II multi-gel casting chamber, or PROTEAN® Plus multi-casting chamber.
  • Use only high-quality reagents, especially acrylamide monomers, to avoid polymerization problems.
  • Proper degassing and filtering of the casting solution is critical for both reproducibility of the polymerization (oxygen removal) and the avoidance of problems related to mass spectrometry (keratin contamination).
  • A temperature of 23–25°C is best for degassing and polymerization; equilibrate the stock solutions to room temperature.
  • APS/TEMED-initiated reactions should proceed for at least 2 hr to ensure maximum reproducibility of pore size.
  • Make fresh APS solution every day for best performance.
  • Replace TEMED every three months because it is subject to oxidation, which causes the gradual loss of catalytic activity.
  • The glass plates must be clean and free of chips. Clean glass plates with ethanol and lint-free cloths before use.
  • The height of the stacking gel should be at least 2x the height of the sample in the well. This ensures band sharpness, even for diluted protein samples.
  • Store gels flat in the fridge at 4°C. Do not freeze. Wrap handcast gels tightly in plastic wrap with combs still inserted.
  • Run handcast gels with discontinuous buffer systems right after gel casting because the buffer discontinuity (pH and ionic strength) gradually disappear during storage. SDS-PAGE gels are not stable at pH 8.8 over a longer time period.

For more about acrylamide polymerization, refer to Bio-Rad bulletin 1156.

Products for Handcasting Gels

Premade Buffers and Reagents
Electrophoresis buffers and reagents are available as individual reagents or as premixed gel-casting, sample, and running buffers. Use of commercially prepared, premixed buffers helps save time but also helps to maximize reproducibility, avoid potential mistakes in buffer concentration and standardize electrophoresis runs, as they are made with electrophoresis-purity reagents and are quality controlled for reproducible results. There are no reagents to weigh or filter; just dilute with distilled or deionized water.

AnyGel™ Stands
AnyGel stands provide stabilization and access to gels for casting and sample loading. The clamping mechanism secures gels cassettes vertically without excess pressure. They are available in two sizes, single- and six-row.

AnyGel stands

AnyGel stands

Multi-Casting Chambers
Multi-casting chambers are sued to cast multiple gels of various thicknesses simultaneously. Acrylic blocks act as space fillers when fewer than the maximum number gels are cst. These chambers work in concert with the gradient formers through a bottom filling port to ensure reproducibility. Multi-casting chambers are available for casting gels for the Mini-PROTEAN®, PROTEAN® II, and PROTEAN® Plus systems.

Gradient Formers
Gradient gels have a gradient of acrylamide concentration that increases from top to bottom. To create this gradient, the acrylamide solution must be mixed in a gradient former before being introduced into the gel cassette. Typically, two solutions are prepared: the light solution (equivalent to the lowest %T in the range to be poured) and a heavy solution (equivalent to the maximum %T to be poured). The most common gradient gel contains 4–20% acrylamide; however, the range of acrylamide concentrations should be chosen on the basis of the size of the proteins being separated.

Two gradient formers are available for PAGE systems. Depending on the gel format, prepare either a single gel using the gradient former, or couple the gradient former with a multi-casting chamber for the preparation of up to 12 gels simultaneously:

Multi-casting chambers and gradient formers

Multi-casting chambers and gradient formers.

Troubleshooting

Problem Cause Solution
Leaking during handcasting Chipped glass plates Ensure plates are free of flaws
Spacer plate and short plate not level Ensure plates are aligned correctly
Casting stand gasket dirty, flawed, or worn out Wash gasket if it is dirty. Replace flawed or worn out casting stand gaskets
Poor well formation Incorrect catalyst used Prepare fresh catalyst solution. Increase catalyst concentration of stacking gel to 0.06% APS and 0.12% TEMED
Monomer solution not degassed
(oxygen inhibits polymerization)
Degas monomer solution immediately prior to casting stacking gel
Webbing; excess acrylamide behind the comb
Incorrect catalyst concentration Prepare fresh catalyst solution. Increase catalyst concentration of stacking gel to 0.06% APS and 0.12% TEMED
Pressure cams on casting frame are difficult to close or make noise when closed Powder residue has built up at pivot point of pressure cams Rinse or wipe off powder residue before each use
Gel does not polymerize Too little or too much APS or TEMED Use 0.05% APS and 0.05% TEMED
Failure to degas Degas monomer solutions 10–15 min
Temperature too low Cast at room temperature, warming glass plates if necessary
Poor quality acrylamide or bis Use electrophoresis-grade reagents
Old APS Prepare fresh APS
Swirls in gel Excessive catalysts; polymerization time <10 min Reduce APS and TEMED by 25% each
Gel inhibition; polymerization time >2 hr Increase APS and TEMED by 50%; degas
Gel feels soft Low %T Use different %T
Poor quality acrylamide or bis Use electrophoresis-grade reagents
Too little cross-linker Use correct %C
Gel turns white Bis concentration too high Check solutions or weights
Gel brittle Cross-linker too high Use correct %C
Sample floats out of well Sample not dense enough Include 10% glycerol in sample to make it denser than surrounding buffer
Pipetting, loading error Pipet sample into well slowly. Do not squirt sample quickly into well, as it may bounce off bottom or sides and flow into next well. Do not remove pipet tip from well before last of sample has left tip

Related Content

Literature
Number Description Download
1156 Acrylamide Polymerization — A Practical Approach Click to download
2317 Ready-to-Run Buffers and Solutions Brochure, Rev F Click to download
6201 Handcasting Polyacrylamide Gels Protocol, Rev B Click to download
6199 Buffer Formulations Click to download
6201 Handcasting Polyacrylamide Gels Click to download
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