EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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electro-blotting (western blotting)

Mai 30, 2018

The gel have to be cut from the film support before starting the blotting process, otherwise no current will flow
This job will do GE’s “Film Remover” (GE 18-1013-75), see pictures below.

Now available: EDC’s Gelremover

2DGelBlot#3Klein

GE’s Film-Remover
with a 2DGel sitting on it: The cutting wire waits on the top, clamped tensely on the right side...

2DGelBlot#3bKlein

...then it is drawn and runs over the film-support and underneath the gel...

2DGelBlot#4Klein

...now the gel is already cutted off!

semidryklein

we recommend to do the blot between 2 graphite plates:  “semi-dry blotting”

...and the film
can be removed easily

2DGelBlot#7Klein

Transfer buffers (Discontinuous buffer system)
Anode solution I:
0.3 mol/L Tris (36.3 g), 20% (v/v) methanol200 mL, make up to 1 L with distilled water.
Anode solution II:
25 mmol/L Tris (3.03 g), 20 %(v/v) methanol 200 mL, make up to 1 L with distilled water.
Cathode solution:
40 mmol/L ε-aminocaproic acid (5.2 g), 0.01% (w/v) SDS0.1 g, 20% (v/v) methanol200 mL,
make up to 1L with distilled water.

Blotting procedure
Wet the graphite anode plate (the plate with the red cable) with distilled water, remove the excess water with tissue paper.
Cut the necessary filter papers (6 for the anode I, 3 for anode II, 9 for the cathode) and the blotting membrane to the size of the gel.
Slowly soak 6 filter papers in anode buffer I, and place the stack in the middle of the blotting plate.
Soak the blot membranes in anode buffer II and place them on the stack.
Cut the gel from the film, see above. The film-gel sandwich is still together and is placed flat on the lab-bench.
Slowly soak 3 filter papers in anode buffer II and place the stack on top of the first stack.
Then, with a roller the dry blotting membrane is rolled on the gel.
Turn the sandwich around, so that the support-film is on top.
With a spatula the support-film is carefully lift upward so that...
          Nitrocellulose-Membrane
            ...the gel is remaining on the membrane
          PVDF-Membrane
           ...the gel is detached from the support with a spatula and is falling on the membrane with some crinkles.
              Dunk this sandwich under 100% Methanol and there the gel’s crinkles are evened onto the
              membrane with a spatula
Grasp the membrane and the gel on 2 corners and place this sandwich -membrane down- on the Anode II stack
Slowly soak 9 filter papers in cathode buffer and apply the stack onto the gel.
Apply the cathodal electrode plate and start the electro-transfer (no cooling)

Runnings

10 V

80 mA / cm**2

normal gel: 400 mA

1 h

Staining

Coomassie-stain inside the gel...

Extrakt10Klein
Pfeil

...PVDF-membrane: electroblot and
Fast Green stain

PVDFBlot#10sKlein