SDS-PAGE gel electrophoresis
Lynn Doran, Steven J Burgess
Abstract
SDS-PAGE gel electrophoresis protocol for analyzing samples from plant leaf tissue via immunofluorescence. In this protocol no Coomassie blue is added to samples, the reason is that this interferes with the fluorescent signal during immunoblot. Instead, samples have already been prepared in Laemmli buffer (minus coomassie, protein extraction procedure), the leading edge of samples can be visualized due to the presence of chlorophyll.
Note
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When using 15 well, 0.75 mm comb, try to limit the volume loaded to 10 μL to minimize the risk of spillover of protein between wells.
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Ensure that accurate volume is pipetted by removing sample stuck to the outside of the pipette tip by wiping the tip on the rim of the sample tube to remove any residual liquid.
Literature:
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdfhttps://www.bio-rad.com/webroot/web/pdf/lsr/literature/10026447.pdf
Before start
Extract protein from samples via Leaf Protein Extraction for Immunoblot and determine Protein Concentration using Qubit 4 Fluorometer.
Previously extracted and quantified protein samples can be stored at -20°C.
Steps
Prepare gel tank and buffers
Create a 1X working dilution of Tris/Glycine/SDS buffer (~1L is required per gel tank) by diluting 10X stock 1:10 with distilled H2O.
Carefully remove the comb from the precast gel and the tape across the bottom.
Assemble the Mini-PROTEAN electrophoresis cell and fill the inner chamber with buffer and the outer chamber up to the recommended mark
Wash the wells with running buffer by pipetting up and down
Prepare Samples
In fresh microcentrifuge tubes, create a dilution of each sample using 1x PEB to a concentration of 3 μg /uL of total soluble protein.
Briefly vortex the sample to shear any DNA contamination.
Load 3µL of Chameleon™ Duo Pre-stained Protein Ladder to the first well.
Load 10µL of each sample (30 µg of total soluble protein) per lane.
Running Gel
Run precast gels at 200 V for ~0h 30m 0s or until the samples have reached the end of the gel. In some applications it may be advantageous to run the chlorophyll off the end of the gel to improve fluorescence and signal on the protein of interest.
Equipment
| Value | Label |
|---|---|
| Mini-PROTEAN Tetra Cell | NAME |
| Gel Electrophoresis Tank | TYPE |
| Bio-rad Laboratories | BRAND |
| 1658005EDU | SKU |
Carefully open precast gel case using an opening lever, by inserting where the black arrows indicate on the gel case.
Remove stacking gel with a blade
Proceed either directly to Protein Transfer using Bio-rad TransBlot Turbo or Total Protein Staining.
