Agarose Gel Electrophoresis
By Purbasha Nandi (03/02/2020)
Use horizontal gel, easy to cast and use, no need for stacking portion.
Resolving buffer same as running buffer for horizontal gel.
Make running buffer: (keep a separate portion w/o SDS for dissolving agarose)
Cast Agarose Gels
Dissolve 1.5% SFR Agarose (low melting agarose) for separation in the range of 600-1000 kDa.
Pre-cool running buffer (w SDS) to 4° C.
Dissolve the agarose in running buffer w/o SDS. For denaturing gel, add SDS to the sample buffer and running buffer. Microwave the mixture (avoid bubbles) and cast the gel to the desired width of 4mm, insert the comb carefully.
(Removing the comb while the agarose is still in a molten state and then reinserting often eliminates the formation of bubbles under the teeth of the comb.)
After the agarose has solidified, place the apparatus at 4°C and allow the agarose to age 20 to 30 minutes. The electrophoresis is to be carried out at 4°C. The apparatus may either be placed in a cold room, the electrophoresis buffer can be circulated through a refrigeration unit, or the apparatus can be packed in wet ice.
Overlay the solidified gel with 2 to 3 mm electrophoresis buffer, 4° C.
Remove the sample comb by lifting vertically in one smooth motion. It is helpful to hold the gel down with the gloved fingers of the other hand to keep the gel from being pulled up when the comb is removed.
Prepare and Load the Sample
Dilute the protein samples to twice the desired concentration using distilled water. Immediately add the diluted sample to an equal volume of 2× sample buffer. (Suspend protein samples in 2X sample buffer, 1:1 (v:v))
If denatured proteins are required, incubate at 95°C-100°C for 5 minutes.
Larger amounts of protein can be loaded, but band thickness increases accordingly.
For a 0.8 cm wide well, 25 ml (50 μg total protein) is recommended for a complex mixture, if staining with Coomassie blue, and 1 ml (10 μg total protein) is needed for samples containing one or a few proteins.
Prepare Sample buffer as :
6. Load a sample volume of 10 to 15 µl into the bottom of each sample well using a pipet with a fine tip or equivalent. (Prior to sample loading examine each sample well to ascertain that air bubbles are not trapped in the well.)
Optimal Voltage and Electrophoretic Times
Avoid higher power settings as the heat generated may melt the agarose.
Electrophorese the gel until the tracking dye travels to the bottom of the resolving gel.
Prestained molecular weight markers such as Colored Protein Markers #L77151 can be used to monitor electrophoresis. The gels can be electrophoresed longer, but care should be taken so that smaller proteins do not travel off the gel.
Detection of Proteins in Agarose Gels
Place the container on a shaker with gentle motion during staining and destaining procedures.
Staining proteins with Coomassie® brilliant blue stain:
COOMASSIE® BLUE STAIN SOLUTION DESTAIN SOLUTION
40% Methanol 20% Methanol 10% Glacial Acetic Acid 5% Glacial Acetic Acid 0.25% Coomassie brilliant blue R-250
Room temperature staining:
A 14.5 cm x 16.5 cm, 1 mm thick agarose gel will stain in approximately 1 to 2 hours. • Destain for 1-4 hours with gentle shaking at room temperature:
Overnight staining
Use 0.125% Coomassie blue R-250 with the same concentrations of methanol and acetic acid as in the stain solution.
Destain approximately 4 hours.
Accelerated staining:
Stain gels using standard stain solutions at 50°C.
A 1 mm thick gel takes approximately 1 hour to stain and 1 hour to destain.
Change the destaining solution 1 time.
Agarose gels become softer at 50°C - use a support to transfer between solutions.
Storage:
Do not store agarose gels in destain solution, they may become brittle and fracture.
Store gels in a 5% glycerol solution or dried.
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