Solution preparation

Different Buffer preparation for Gel Electrophoresis

THIS BLOG INCLUDES: hide 1 Acrylamide Stock Solution Preparation 2 Electrode Buffer Preparation 3 Electrophoretic Transfer Buffer (20x) Preparation 4 Imidazole-SDS Solution...

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SouravBio
This article writter by SouravBio on June 13, 2021

Writer and Founder of Microbiologynote.com. I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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Buffer preparation
Buffer preparation

Acrylamide Stock Solution Preparation

Application: Acrylamide is used along with bisacrylamide to form gels used in SDS-PAGE. Acrylamide forms polyacrylamide polymers, while bisacrylamide crosslinks between polyacrylamide chains.

To prepare 1L of Acrylamide Stock Solution the following components are required;

ComponentAmountConcentration
acrylamide (mw: 71.08 g/mol)389.6 g5.481 M
bisacrylamide (mw: 154.17 g/mol)10.4 g0.0675 M

Acrylamide Stock Solution Preparation Steps

  1. Prepare 800 mL of distilled water in a suitable container.
  2. Add 389.6 g of acrylamide to the solution.
  3. Add 10.4 g of bisacrylamide to the solution.
  4. Add distilled water until volume is 1 L.
  5. Filter the stock solution using Whatman filter paper and store at 4°C. Prepare fresh acrylamide stock solution every few weeks. T denotes the total percentage concentration of the monomers (acrylamide plus N,N’-methylenebisacrylamide) in grams per 100 mL. C denotes the percentage (by weight) of the cross-linker N,N’-methylenebisacrylamide relative to the total amount of monomers.

Electrode Buffer Preparation

Application: Electrode buffer has been used in studies involving polymer solar cells.

To prepare 1L of Electrode Buffer the following components are required;

ComponentAmountConcentration
Tris base (mw: 121.14 g/mol)11.6 g0.096 M
glycine (mw: 75.07 g/mol)37.5 g0.5 M
SDS (mw: 288.38 g/mol)3.98 g0.0138 M

Electrode Buffer Preparation Steps

  1. Prepare 800 mL of distilled water in a suitable container.
  2. Add 11.6 g of Tris base to the solution.
  3. Add 37.5 g of glycine to the solution.
  4. Add 3.98 g of SDS to the solution.
  5. Add distilled water until volume is 1 L.

Electrophoretic Transfer Buffer (20x) Preparation 

Application: Electrophoretic Transfer Buffers are used in SDS-PAGE to transfer proteins from a gel to a nitrocellulose membrane.

To prepare 1L of Electrophoretic Transfer Buffer (20x) the following components are required;

ComponentAmountConcentration
Trisodium citrate (mw: 258.07 g/mol)35.29 g0.12 M
Sodium phosphate, dibasic (Na2HPO4) (mw: 141.96 g/mol)22.71 g0.16 M

Electrophoretic Transfer Buffer (20x) Preparation Steps

  1. Prepare 800 mL of distilled water in a suitable container.
  2. Add 35.29 g of Trisodium citrate to the solution.
  3. Add 22.71 g of Sodium phosphate, dibasic (Na2HPO4) to the solution.
  4. Add distilled water until volume is 1 L.
  5. Adjust the pH to 8.3, autoclave, and store for up to 1-2 yr at room temperature.

Imidazole-SDS Solution Preparation

 To prepare 1L of Imidazole-SDS Solution the following components are required;

ComponentAmountConcentration
imidazole (mw: 68.08 g/mol)13.6 g0.2 M
SDS (mw: 288.38 g/mol)1 g0.0035 M

Imidazole-SDS Solution Preparation Steps

  1. Prepare 800 mL of distilled water in a suitable container.
  2. Add 13.6 g of imidazole to the solution.
  3. Add 1 g of SDS to the solution.
  4. Add distilled water until volume is 1 L.

SDS-PAGE SDS Running Buffer (10x) Preparation

Application: In SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), SDS Running Buffer is used as the electrophoresis buffer during stacking and resolution.

 To prepare 1L of SDS-PAGE SDS Running Buffer (10x) the following components are required;

ComponentAmountConcentration
Tris base (mw: 121.14 g/mol)30.3 g0.2501 M
Glycine (mw: 75.07 g/mol)144.4 g1.924 M
SDS (mw: 288.38 g/mol)10 g0.0347 M

SDS-PAGE SDS Running Buffer (10x) Preparation Steps

  1. Prepare 800 mL of distilled water in a suitable container.
  2. Add 30.3 g of Tris base to the solution.
  3. Add 144.4 g of Glycine to the solution.
  4. Add 10 g of SDS to the solution.
  5. Add distilled water until volume is 1 L.

Sodium Carbonate Transfer Buffer (40x, pH 9.5) Preparation

Application: Sodium Carbonate Transfer Buffer is used to transfer proteins following SDS-PAGE.

To prepare 1L of Sodium Carbonate Transfer Buffer (40x, pH 9.5) the following components are required;

ComponentAmountConcentration
NaHCO3 (mw: 84.01 g/mol)21.1 g0.251 M
Na2CO3 (mw: 105.99 g/mol)18.35 g0.173 M

Sodium Carbonate Transfer Buffer (40x, pH 9.5) Preparation Steps

  1. Prepare 800 mL of distilled water in a suitable container.
  2. Add 21.1 g of NaHCO3 to the solution.
  3. Add 18.35 g of Na2CO3 to the solution.
  4. Adjust the pH to 9.5 with 10 N NaOH.
  5. Add distilled water until volume is 1 L.
  6. Store at room temperature.

Tris-Glycine Transfer Buffer (20x) Preparation 

Application: Tris-Glycine Transfer Buffer is used as a transfer buffer in Western blotting.

To prepare 1L of Tris-Glycine Transfer Buffer (20x) the following components are required;

ComponentAmountConcentration
Tris base (mw: 121.14 g/mol)24.2 g0.2 M
Glycine (mw: 75.07 g/mol)150.1 g2 M

Tris-Glycine Transfer Buffer (20x) Preparation Steps

  1. Prepare 800 mL of distilled water in a suitable container.
  2. Add 24.2 g of Tris base to the solution.
  3. Add 150.1 g of Glycine to the solution.
  4. Add distilled water until volume is 1 L.
  5. pH adjustment is not necessary (it will be ~8.8). Store at room temperature.

Tris/EACA Buffer Preparation

To prepare 1L of Tris/EACA Buffer Preparation the following components are required;

ComponentAmountConcentration
ε-aminocaproic acid (EACA) (mw: 131.17 g/mol)1.31 g0.01 M
Tris base (mw: 121.14 g/mol)10.9 g0.09 M

Tris/EACA Buffer Preparation Steps

  1. Prepare 800 mL of distilled water in a suitable container.
  2. Add 1.31 g of ε-aminocaproic acid (EACA) to the solution.
  3. Add 10.9 g of Tris base to the solution.
  4. Add distilled water until volume is 1 L.

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Writer and Founder of Microbiologynote.com. I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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