Creativity

Innovation

Originality

Imagination

 

Salient

Salient is an excellent design with a fresh approach for the ever-changing Web. Integrated with Gantry 5, it is infinitely customizable, incredibly powerful, and remarkably simple.

Download

Protein Blotting Techniques

Membrane Preparation

PVDF membrane

  1. Immerse the membrane in 100% methanol for a few seconds, until the entire membrane is translucent.
  2. Rinse the membrane in deionized water.
  3. Transfer the membrane to transfer buffer, and incubate until it is equilibrated (2-3 minutes or until it no longer floats). Do not allow the membrane to dry.

PVDF wet-blotting with Bio-Rad device

  1. Pre-chill 900 ml of transfer buffer
  2. Prepare the gel sandwich in the cassette, whith the gray side facing down. All components should be soaked in transfer buffer. After addition of each component, roll out bubbles using a pipette. Add in the following order:
    • Fiber pad
    • Whatman paper
    • Gel
    • PVDF membrane
    • Whatman paper
    • Fiber pad
  3. Place the cassette in the holder, and the holder in the tank. Add the cooling block, and fill with blotting buffer to the mark. Remember, proteins run to the positive (red) pole. Use a stir bar to maintain an even ion distribution and for better heat distribution.
  4. Run the blot at 100V, with a 350 mA maximum for 1 hour, or 30V, with a 90 mA maximum overnight.

Transfer buffer:

  • 25 mM Tris
  • 192 mM glycine
  • 20% v/v methanol
  • pH 8.3

PVDF semi-dry blotting with Bio-Rad Trans-Blot device

  1. Pre-chill 100 ml of transfer buffer.
  2. Assemble the stack on the bottom (anode) plate as follows:
    • Two sheets of whatman wetted with transfer buffer.
    • PVDF membrane, prepared as described above.
    • The gel.
    • Two more sheets of whatman wetted with transfer buffer.
  3. Place the cathode and the top cover onto the stack.
  4. Transfer for 15-30 minutes at 10-15V for mini-gels.
  5. After transfer, rinse the membrane three times (5 minutes each) with distilled water.

PVDF wet-blotting with Invitrogen device

  1. Prepare 500ml of NuPAGE transfer buffer from the 20x stock:
    • 25 ml NuPAGE 20x buffer
    • 50 ml methanol
    • 0.5 ml NuPAGE Antioxidant
    • Deionized water to 500 ml
  2. Assemble the transfer stack as follows in the deep (anode) compartment (all components should be soaked in transfer buffer):
    • 3 blotting pads
    • 2 layers of whatman
    • The gel
    • PVDF membrane, prepared as described above.
    • 2 layers of whatman
    • 3 blotting pads
  3. Put the top plate on, and slide the assembly into the buffer chamber. Then lock the tension wedge.
  4. Fill the blot module with transfer buffer to the top of the blotting pads.
  5. Fill the outside of the chamber with room temperature water.
  6. Run at 30 Volts for 1 hour.
Transfer buffer 1
(proteins 20,000-400,000 kDa):

50 mM Tris
380 mM Glycine
0.1% SDS
20% Methanol
H2O

5.8 g
29 g
1 g
200 ml
to 1L

Do not adjust the pH.
Transfer buffer 2
(proteins <80,000 kDa):
25 mM Tris
190 mM Glycine
20% Methanol
H2O
2.9 g
14.5 g
200 ml
to 1L
Do not adjust the pH.
NuPage transfer buffer (20x)
25 mM Bicine
25 mM Bis-Tris (free base)
1 mM EDTA
H2O
81.6 g
104.8 g
6 g
to 1L
pH should be 7.2, do not adjust.
Store at 4°C, stable for 6 months.