Learn about the reagents and techniques and examples of use for electrophoresis. Electrophoresis is a technique which separates charged biomolecules based on the rate at which they migrate in an applied electrical field. In many cases, electrophoresis of proteins are performed using polyacrylamide gel electrophoresis (PAGE). For molecular weight estimation and purity determination of proteins, sodium dodecyl sulfate (SDS)-PAGE is frequently employed. Laemmli’s method is the most widely used system of SDS-PAGE. Agarose gel electrophoresis is one of the most common methods used to size-separate and analyze DNA.

1. Electrophoresis Reagents

2. Electrophoresis
• Electrophoresis is a technique which separates charged biomolecules
based on the rate at which they migrate in an applied electrical field.
• In many cases, electrophoresis of proteins are performed using
polyacrylamide gel electrophoresis (PAGE). For molecular weight
estimation and purity determination of proteins, sodium dodecyl
sulfate (SDS)-PAGE is frequently employed.
• Agarose gel electrophoresis is one of the most common methods
used to size-separate and analyze DNA.

3. Reagents for Gel Preparation, Buffer Preparation, etc
A3217 30% Acrylamide / Bis-acrylamide (29:1) [for Electrophoresis]
A3218 30% Acrylamide / Bis-acrylamide (37.5:1) [for Electrophoresis]
A1132 Acrylamide Monomer [for Electrophoresis]
M0506 N,N'-Methylenebisacrylamide [for Electrophoresis]
A2098 Ammonium Peroxodisulfate [for Electrophoresis]
B3195 Bromophenol Blue Sodium Salt (= BPB) [for Electrophoresis]
D3647 DL-Dithiothreitol (= DL-DTT) [for Electrophoresis]
G0316 Glycerol [for Electrophoresis]
G0317 Glycine [for Electrophoresis]
M1948 2-Mercaptoethanol [for Electrophoresis]
S0588 Sodium Dodecyl Sulfate (= SDS) [for Electrophoresis]
T2515 N,N,N',N'-Tetramethylethylenediamine (= TEMED) [for Electrophoresis]
T2516 Tris(hydroxymethyl)aminomethane (= Tris-Base) [for Electrophoresis]
T3843 1-Thioglycerol [for Electrophoresis]

4. Protein Electrophoresis- SDS
• SDS is a strong denaturant of proteins and is added to samples, gels,
and buffer solutions for electrodes when proteins are separated with
electrophoresis.
• As SDS not only denatures protein but also binds to the protein, when
SDS is used in conjunction with a reducing reagent such as 2-
mercaptoethanol to cleave disulfide bonds in the protein, and the
protein is completely denatured, the amount of SDS bound is almost
always proportional to the molecular weight of the protein.
• Resultantly, the protein is negatively charged. Therefore, the
denatured protein can be separated by molecular weight
independently of its structure and biological properties.

5. Laemmli’s Method - SDS
• Laemmli’s method is the most widely used system of SDS-PAGE. In
this method, the separation and the stacking gel contain Tris-HCl and
the upper and lower buffer reservoirs contain Tris-glycine. All
components of the system contain SDS.
• The advantage of Laemmli’s method is that it gives sharper bands in
the final plate.

6. SDS-PAGE Sample Buffers
• Sample buffers are available in three different concentrations to allow
for easy use with any sample volume. No reducing agent is included-
add as required. A red sample bufferis also available to help prevent
sample mix-up.
• For Example of use, Visit:
https://www.tcichemicals.com/IN/en/product/topics/electrophoresis_reagents
B5834 2X SDS-PAGE Sample Buffer (2-Mercaptoethanol free) [for Electrophoresis]
B6104 4X SDS-PAGE Sample Buffer (2-Mercaptoethanol free) [for Electrophoresis]
B6105 6X Sample Buffer (2-Mercaptoethanol free) [for Electrophoresis]
B6110 2X SDS-PAGE Sample Buffer Phenol Red (2-Mercaptoethanol free) [for Electrophoresis]

7. Gel Staining Reagent
• Detecting protein bands in a polyacrylamide gel after electrophoresis
requires the gel to be stained. Common staining methods include
Coomassie Brilliant Blue staining, silver staining, fluorescence
staining, and negative staining.
• Negative staining is a method in which only regions of gel containing
protein remain unstained, while the remainder of the gel is stained
white.
• Bands can be visualized by placing the gel against a dark background.

8. Gel Negative-Staining Kit
• The Gel Negative-Staining Kit is used for Rapid and Highly Sensitive
Protein Detection in as little as 20 minutes.
• Furthermore, stained gels can easily be destained using the included
destaining solution, with the destained gel is able to be used in
further downstream applications, such as Western blotting and mass
spectrometry.
G0615 Gel Negative Stain kit

9. Example of use (Staining gels with G0615)
Figure. Photograph of gel stained by G0615
1) Place the post-SDS-PAGE gel in a tray containing
enough deionized water to cover the gel and shake
for 10 minutes.
2) Discard the deionized water, add enough
solution A (diluted 10 times with deionized water)
to cover the gel and shake for 5 minutes.
3) Submerge gel in deionized water for 10 seconds
to wash. Repeat three times.
4) Transfer the gel to a new tray, add enough
solution B (diluted 10 times with deionized water)
to cover the gel and shake for 1 minute to develop
color.

10. Example of use (Preparation of gel for Western
Blotting)
• Place the stained and photographed gel
in a tray containing solution C diluted
10-fold with deionized water.
• Shake the gel until the color is removed.
• Discard solution C, add enough
deionized water to cover the gel and
wash for 30 seconds. Repeat three
times.
• Transfer the washed gel to a membrane
(PVDF).
Figure. A: Gels loaded with mouse serum and stained with G0615.
B: The gel in A was destained and mouse IgG was detected via Western Blotting.

11. Gel Staining Reagent (Methanol-free CBB Stain
Solution)
C3488 is a methanol- and acetic acid-free one-component ready-to-use
solution for staining proteins.
Example of use (Staining gels with C3488)
1) After gelelectrophoresis, wash the gel with deionized water for 5 minutes
three times.
2) Remove the water, add C3488 till the gel is soaked and shake the gel
gently for 1 hour at room temperature.
3) Remove C3488 and destain the gel with deionized water for 1 hour. If high
background staining is observed, destain the gel with deionized water
overnight at room temperature.
C3488 Coomassie Brilliant Blue G-250 (Ready-to-use solution) [for Electrophoresis]

12. Figure. Proteins stained by C3488 (destained overnight)

13. Reagents for Protein Staining and Others
A2097 Acid Black 1 (= Amido Black 10B) [for Electrophoresis]
A2256 Acid Red 112 (= Ponceau S) [for Biochemical Research]
B3193 Coomassie Brilliant Blue G-250 [for Electrophoresis]
B3194 Coomassie Brilliant Blue R-250 [for Electrophoresis]
F0718 Fast Green FCF [for Biochemical Research]
D1820 Sodium Deoxycholate [for Electrophoresis]
A2255 6-Aminohexanoic Acid [for Biochemical Research]

14. DNA Electrophoresis
• The nucleotides that make up DNA carry a negative charge due to
their phosphate groups, and are therefore attracted to the anode
when run on an agarose gel.
• As the DNA moves through the gaps in the mesh of the agarose gel,
longer molecules, with higher molecular weights, move more slowly,
while smaller molecules are able to move more quickly.
• This method, of separating molecules by size on a gel using electricity,
is known as gel electrophoresis.

15. Nucleic Acid Staining Reagent (Ethidium Bromide Solution)
L0393 6X Loading Buffer Bromophenol Blue [for Electrophoresis] [for Molecular Biology] (1 mL×3)
L0440 6X Loading Buffer Double BX [for Electrophoresis] [for Molecular Biology] (1 mL×3)
Nucleic Acid Sample Preparation Reagents for Electrophoresis
Ethidium bromide is widely used as a DNA staining agent with agarose gels and
can detect DNA with high sensitivity through visualization using UV
light. However, its carcinogenicity means it must be handled with care.
E1363 comes in a dropper bottle pre-dissolved, making it safe and easy to use.
E1363 Ethidium Bromide (0.5mg/mL in Water) (in Dropper Bottle) [for Electrophoresis]

16. Example of use (Staining gels with E1363)
1) After electrophoresis, dilute E1363 (1
drop / 40 mL) to 0.5 µg/mL with water or
running buffer and incubate the gel for 15
minutes.
2) If you have to decrease background
fluorescence, wash the gel in water for 15
minutes.
3) In use of electrophoresis buffer
solution, Ethidium bromide incorporated
into nucleic acid and can visualize band
immediately by using UV transilluminator.
Figure. DNA Ladder Marker stained by E1363 (destained 15 minutes)

17. Nucleic Acid Staining Reagent (Nucleic Acid
Stain Blue)
Nucleic acid stain blue is used to stain nucleic acids after agarose gel
electrophoresis. As nucleic acids are stained blue, no transilluminator
or other detection device is required. Unlike ethidium bromide, it is
non-mutagenic and therefore safe and easy to handle.
N1209 10X Nucleic Acid Stain Blue [for Electrophoresis]

18. Example of use (Staining gels with N1209)
1) Prepare N1209 diluted 10 times with
deionized water.
2) After electrophoresis, immerse the
gel in the diluted N1209 and shake
for 10 minutes.
3) Discard the staining solution and
shake the gel with deionized water
for 30 minutes and wash the gel.
Repeat the wash step if background
is high.
Figure. Photograph of gel stained by N1209 (destained overnight)