TITLE: SDS-PAGE gel electrophoresis
AUTHORS: Steven Burgess

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.  </div><div class = "text-block"><span style = "font-weight:bold;">Note </span></div><div class = "text-block">- 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. </div><div class = "text-block">- Ensure to wipe the tip on the rim of the sample tube to remove the sample stuck to theoutside of the tip. </div><div class = "text-block"><span style = "font-weight:bold;">Literature: </span></div><div class = "text-block"><a href="http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf</span></a><a href="https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10026447.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10026447.pdf</span></a></div></div>

[STEPS]
?. [Prepare gel tank and buffers]
Create a working dilution of Tris-Glycine running buffer (~  is required per gel tank) by diluting 1:10 with d H2O.
1 L
?. [Prepare gel tank and buffers]
Carefully remove the comb from the precast gel and the tape across the bottom.
?. [Prepare gel tank and buffers]
Assemble the Mini-PROTEAN electrophoresis cell and fill the inner chamber with buffer and the outer chamber up to the recommended mark
the volume varies depending on whether running 2 or 4 gels, the level is marked on the tank
?. [Prepare gel tank and buffers]
Wash the wells with running buffer by pipetting up and down
This is done to remove residual acrylamide that may have collected in wells
?. [Prepare Samples]
In fresh centrifuge tubes, create a dilution of each sample using 1x PEB, such that each sample is set at a concentration of 3 μg /mL of total soluble protein.
Recommended final volume ~  (this will allow for 10 samples) but will depend on the application
?. [Prepare Samples]
Heat samples at  for
95 °C
?. [Prepare Samples]
Spin down samples at  for  to pellet debris
Centrifuge: 10000 34
?. [Prepare Samples]
Load  of Chameleon™ Duo Pre-stained Protein Ladder to the first well
3 µl
?. [Prepare Samples]
Load  of each sample (30 µg of total soluble protein) per lane
10 µl
?. [Running Gel]
Run precast gels at 200 V for ~ .
Or until the samples have reached the end of the gel. For self-made gels, run at 80-120 V
?. [Running Gel]
Carefully open precast gel case using an opening lever, by inserting where the black arrows indicate on the gel case.
?. [Running Gel]
Remove stacking gel with a blade
?. [Running Gel]
Proceed either directly to protein transferor if the gel is as a loading control, place in a container and cover with .
{"blocks":[{"key":"3s7ks","text":"This protocol is based on the assumption that TGX pre-cast gels have been used, the procedure is very similar for self-made gels, just that the running time is longer. Using the transfer packs is economically similar to wet-transfer using MeOH.  ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"akb94","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"6bldi","text":"Note:  Clean the apparatus immediately after use with dH2O and dry. This prevents the build-up of salts and rust which impair performance.  ","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":4,"offset":0,"style":"bold"},{"length":1,"offset":56,"style":"sub"}],"entityRanges":[],"data":[]},{"key":"11gqu","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"ecfr7","text":"Literature: ","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":10,"offset":0,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"93p0l","text":"Full manual: https:\/\/www.bio-rad.com\/webroot\/web\/pdf\/lsr\/literature\/10020688.pdf ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[{"key":0,"length":67,"offset":13}],"data":[]},{"key":"9tjr3","text":"Quick-start: https:\/\/www.bio-rad.com\/webroot\/web\/pdf\/lsr\/literature\/10016505D.pdf ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[{"key":1,"length":68,"offset":13}],"data":[]},{"key":"98u0s","text":"Transfer pack: https:\/\/www.bio-rad.com\/webroot\/web\/pdf\/lsr\/literature\/10019593D.pdf","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[{"key":2,"length":68,"offset":15}],"data":[]}],"entityMap":[{"type":"link","data":{"guid":"1397388238EC11EBA61A0A58A9FEAC2A","url":"https:\/\/www.bio-rad.com\/webroot\/web\/pdf\/lsr\/literature\/10020688.pdf"}},{"type":"link","data":{"guid":"139738FE38EC11EBA61A0A58A9FEAC2A","url":"https:\/\/www.bio-rad.com\/webroot\/web\/pdf\/lsr\/literature\/10016505D.pdf"}},{"type":"link","data":{"guid":"1397395C38EC11EBA61A0A58A9FEAC2A","url":"https:\/\/www.bio-rad.com\/webroot\/web\/pdf\/lsr\/literature\/10019593D.pdf"}}]}
?. [Visualizing Gel (optional)]
Gently agitate the gel on a rocking platform for .
?. [Visualizing Gel (optional)]
Pour off the Imperial Protein Stain (collect as hazardous waste)
?. [Visualizing Gel (optional)]
Rinse the gel with dH2O.
?. [Visualizing Gel (optional)]
Cover the stained gel with dH2O and gently agitate in a rocking platform for
?. Repeat steps  15-17 until the background of the gel is clear and blue protein bands can be clearly visualized
?. Image the gel on the Bio-Rad Gel Doc XR system trans-white illumination