Electrophoresis Protocols

ProtoGel (40%) Protocol

Measure and Mix Solutions:
The volume of ProtoGel required for gel casting solutions of any volume and acrylamide concentration may be calculated from the following formula:
Vp =(X) (Vt)/40
where:
Vp = Volume of 40% ProtoGel
X = % Monomer Desired in Gel
Vt = Total Volume of Gel Casting Solution

EXAMPLE: To make 100 ml of a 10% monomer gel, calculate the volume of Protogel to add as follows:
Vp = (10) (100)/40 = 25.0 ml

ProtoGel (30%) Protocol

Measure and Mix Solutions:
The volume of ProtoGel required for gel casting solutions of any volume and acrylamide concentration may be calculated from the following formula:
Vp =(X) (Vt)/30
where:
Vp = Volume of 30% ProtoGel
X = % Monomer Desired in Gel
Vt = Total Volume of Gel Casting Solution

EXAMPLE: To make 100 ml of a 10% monomer gel, calculate the volume of Protogel to add as follows:
Vp = (10) (100)/30 = 33.3 ml

SequaGel XR Protocol

Mix Solutions
Add appropriate volumes of SequaGel XR Monomer Solution and SequaGel Complete Buffer to a thick-walled Erlenmeyer flask (see Table 1). If desired, the solution may be degassed by stirring under vacuum for two minutes.
Bring to room temperature before polymerization.

Table 1: Volumes of SequaGel XR Monomer Solution and
SequaGel Complete Buffer to prepare 100mL gel solution

UreaGel 6 and 8 Protocol

Mix Solutions
Add appropriate volumes of SequaGel 6 or 8 Monomer Solution and SequaGel Complete Buffer to a thick-walled Erlenmeyer flask (see Table 1). If desired, the solution may be degassed by stirring under vacuum for two minutes.
Bring to room temperature before polymerization.

Table 1: Volumes of SequaGel 6 or 8 Monomer Solution and
SequaGel Complete Buffer to prepare 100mL gel solution

SequaGel UreaGel System Protocol

Mix UreaGel System Components:
Determine how much UreaGel Concentrate, Diluent, and Buffer you need to make your gels using the formulas below. Combine the necessary components in an Erlenmeyer flask. Swirl gently to mix.

For 100 ml Gel Casting Solution:
Vc =(Vt) (X)/25
Vb = 0.1 (Vt)
Vd = Vt - (Vc + Vb)

AcrylaGel Protocol

Calculate how much AcrylaGel and Bis-AcrylaGel you need to make your gels by using the formulas below. Bring up to the desired final volume with your usual buffers and distilled water.


Mix Gel Solution
Va = (A)(Vt)/30

Vb = (A)(C)(Vt)/200
Va = Volume of AcrylaGel to be used (ml),
Vb = Volume of Bis-AcrylaGel to be used (ml),
Vt = Total volume of gel casting solution desired (ml),
A = % acrylamide desired in gel,
C = % crosslinker desired

NucliStain Protocol

  1. To prepare a working strength solution of NucliStain, add one part NucliStain concentrate to one-hundred parts of distilled water. Mix until the solution is uniform. Prepare sufficient stain solution to fully submerge the gel.
  2. Immerse the gel in the stain solution for 20–30 minutes.
  3. Immerse the gel in distilled water for 3–4 hours to destain the blue background. Bands can often be visualized minutes after destaining. However, for increased sensitivity, overnight destaining is recommended.

ProtoBlue Safe Protocol

Prepare Working Solution
1. Gently invert the bottle several times to resuspend colloidal dye particles that settle out on standing.
2. Add 1 part ethanol to 9 parts staining solution while stirring. (Standard denatured ethanol is fine). A 20ml to 50ml volume of working solution is typically prepared, depending on the shape and size of the staining container. To preserve solution, we recommend a plastic staining container just big enough to hold the gel.

Procedures for Gel Staining

Sterling Silver Stain Protocol

EASY AS 1-2-3
1. FIX gel for at least 25 minutes in 100ml of the standard mixture of 5:5:1 Methanol:Water:Acetic Acid. Decant fixative, then add reconstituted STERLING Fixative (45ml water, 50ml methanol, 5ml STERLING Fixative Concentrate) and fix for an additional 5 minutes
2. WASH gel twice for 15 minutes in deionized water. Addition of 0.1% non-ionic surfactant will aid in submerging the gel.

     • While gel is washing, prepare Staining Solution as directed. Do not combine the two component solutions until just prior to use.

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