Re-using plastic cassettes for vertical gel electrophoresis.

doi:10.1038/protex.2016.041

Method Article

Re-using plastic cassettes for vertical gel electrophoresis.

https://doi.org/10.1038/protex.2016.041

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Vertical gel electrophoresis is frequently used for separating proteins via SDS-polyacrylamide electrophoresis (SDS-PAGE) or for separating DNA or RNA in polyacrylamide gels. Some vertical gel electrophoresis units do not allow the usage of glass plates, instead plastic cassettes must be used. These cassettes are only for single usage as the disassembly involves the irreversible separation of the gel plates. Here we report on a procedure that allows the multiple usage of such plastic gel cassettes. This procedure can be used for any electrophoresis unit that applies pressure on the mounted plastic gel cassette such that it presses the gel plates together, for example the NOVEX system (Thermo Fisher Scientific). [*These authors contributed equally.]

Biochemistry

Biological techniques

Molecular Biology

Computational biology and bioinformatics

SDS-PAGE

Acrylamide gel

Electrophoresis

• 70 % ethanol

• 1 % agarose in deionised or distilled H₂O, microwave to liquefy

• 4 small foldback clips (32 mm or 1-1/2"), or 3 larger foldback clips (41 mm or 1-5/8"), see Figure 5A & 5B.

• One set of plastic gel plates, tall and short,

Important: Gel plates must originate from the same plastic cassette.

• Paper towels or Whatman filter paper

• Scotch tape or masking tape

• Comb

• Microwave

Clean plastic gel plates with 70 % ethanol. Do not use acetone as it damages the plastic (Fig. 1A).
Place the tall plate on the lab bench, alternatively place tall plate on a yellow-pipette-tip box as shown in Figure 2B. Pipette 2-3 ml of 1 % liquefied agarose along the outside part of the plate. The agar must cover well the outside part (Fig. 1B).
Carefully place the short plate onto the tall plate (Fig 1C).
Carefully lift up the cassette, while holding it levelled apply the foldback clips as shown in the figure (Fig. 2A). Then place assembled cassette on the bench horizontally. If you have placed the gel plate on a pipette tip box, the clips can be applied without lifting the plate (Fig. 2B). The foldback clips should apply pressure on the tall plate’s rim that gets in contact with the short plate (Fig 2C).
After the agarose has solidified, lift up the cassette and check whether the outside part of the cassette is well filled with agarose. If this is not the case, pipet from the outside agarose in-between the plates, then re-apply the foldback clips, and let the agarose solidify (Fig. 3A)
If agarose has leaked into the inside of the cassette, use a piece of a paper towel or Whatman Blotting paper to remove the agarose (Fig. 3B)
Tape scotch tape over the slot at the bottom of the tall gel plate (Fig. 4). Note: Folding over one end of the tape will allow easier removal later-on.
The cassette is now ready for casting a gel (Fig. 5A or 5B). For pouring an SDS-PAGE gradient gel see reference (1), but use 7.5 ml of each premix acrylamide solution, and 75 µl of APS and 7.5 µl of TEMED per mixing chamber.
After the acrylamide gel has solidified, remove the comb and scotch tape. Carefully remove the foldback clips, rinse with deionised water, remove excess water, and carefully mount the gel cassette into the electrophoresis unit. (Fig. 6)
Load samples following usual procedures.

Approximately 10 min

When loading samples they do not stay in the gel pockets, instead they travel downwards between the gel and the gel plate.

• It is important to use gel plates that originated from the same gel cassette. This is due to the nature of the plastic pins connecting the two plates in the original cassette. When separating the plates these pins break apart differently in each cassette.

The cassette is leaking when casting the acrylamide gel.

• Make sure the agarose has fully sealed the gap between the plates (on the outside of the cassette) (Fig. 2C). If necessary fill up gap with agarose (Fig. 3A).

• Make sure that the tall and short plates are secured with foldback clips before the agarose solidifies. Do not move the cassette when the agarose has not solidified yet.

• Make sure the tape is covering the slot well. Press the tape on well, and make sure the tape adheres well to the plastic all around the slot

• Leakage is usually not a problem. However, if necessary, to check whether the assembly does not leak, fill the cassette with water and check for leakage. Then pour out the water, and insert parts of a paper towel or Whatman Blotting paper, to remove any remaining water.

During electrophoresis the samples move towards the left and right side of the gel rather than move straight down.

• This is likely due to the current not only going from top to bottom of the gel but also through the sides of the gel

• Make sure you use 1% agarose made in deionised or distilled water, but not in buffer

• Fill just enough running buffer into the bottom chamber. If the bottom chamber must be filled up all the way, then apply plastic scotch tape along the left and right side of the cassette to prevent current to enter through the sides of the cassette.

The slots contain pieces of agar, preventing proper loading of samples

• The comb thickness may not match the void between the plates (i.e. the thickness of the comb is less that the gap between the plates). In this case a thin layer of acrylamide polymerises in the space between the comb and the plate. Once the comb is removed, this layer of acrylamide usually tears and settles in the wells, cannot be removed, and affects sample loading.

• If the comb has the proper thickness, and problems still occur, secure the comb with a foldback clip immediately after pouring the gel.

With this procedure, plastic gel cassettes can be used >20 times for casting acrylamide gels.

(1) Simultaneous semi-dry electrophoretic transfer of a wide range of differently sized proteins for immunoblotting. Martin Wiedemann, Su Jung Lee, Richard Cardoso da Silva, Jyothsna Visweswaraiah, Josefin Soppert, and Evelyn Sattlegger, Protocol Exchange, 18/12/2013, doi:10.1038/protex.2013.095

The authors of this protocol are:

Rashmi Ramesh, Jan Gebler, Valerie de Boor & Evelyn Sattlegger

The authors declare no competing financial interests.

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Re-using plastic cassettes for vertical gel electrophoresis.

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Re-using plastic cassettes for vertical gel electrophoresis.

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Abstract

Figures

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

References

Additional Declarations

Associated Publications

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Terms of Service

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