1

Soak 1 stack of transfer stacks in 35 ml Blot buffer.

2

Place the transfer stack in the Turbo-Blot tray.

3

Keep the membrane between the blue papers as much as possible to avoid protein contamination. Do not touch the membranes with bare hands: wear gloves and use tweezers.

• Cut the membrane in half, so that you have one membrane for each gel. (check with the assistant how!)
• Mark the membranes with pencil to distinguish them from each other and from the membranes of other teams. Write antibody name + team number on the bottom corner.

4

Do not touch the membranes with bare hands: wear gloves and use tweezers.

• Activate the PVDF membranes by placing the membranes in 100% EtOH for 2-3 minutes. (Don’t forget to remove the blue papers first)
• Place the PVDF activated membrane on the transfer stacks with the labeled side up.

5

Carefully remove the top glass plate from the gel.

6

Remove the stacking gel (the part of the gel where the slots are) and gently place the gel on the membrane.

Repeat this step for the 2^nd gel.

7

• Soak the 2^nd stack of transfer stacks in 35 ml Blot buffer.
• Place the stack of transfer stacks on top of the gels.
• Close the tray by putting the lid.

8

Start the 7-minute transfer program.

9

Remove the tray from the machine and check if you can see the protein ladder on the membrane (NOTE: On which side are your proteins ?!)

What is the correct way to assemble a transfer stack? Start with the tray block on top, and add the others in the right order downwards.

• Do you have your tray block on top?
• Think in which direction the proteins move.

1. a Transfer stack
2. b Transfer stack
3. c Gel
4. d PVDF membrane
5. e Lid
6. f Tray

• Correct answer fadcbe or fbdcae
  Yes! this is the correct order
• Incorrect answer
  Your order is not correct.

Now you know how to build up your transfer stack and you know how the charge has influence on the process, can you explain where the positive and negative pool are located?

Proteins are [a].

This means the tray contains the [b] pool, and the lid contains the [c] pool.

1. Option: Positive
2. Option: Negative
3. Very good! Proteins are negative. This means they run to the positive pool. Since they have to transfer from the gel to the membrane, this means the positive pool is in the tray. Have a look the transfer stack order again.

• Incorrect answer a
  Your protein charge is not correct.
  What is the direction of electricity?
  Proteins are negative. So do they run to the positive or negative pool? Check with your transfer stack.

Wat gebeurt er bij activatie van het PVDF membraan?

1. Het hydrofiele membraan wordt door toevoegen van water geactiveerd. Hierdoor verkrijgt het membraan de volle eiwit binding capaciteit.
   • Incorrect. Is er een hydrofiel of een hydrofoob membraan nodig om eiwitten te binden ?
2. Het hydrofiele membraan wordt door toevoegen van methanol geactiveerd. Hierdoor verkrijgt het membraan de volle eiwit binding capaciteit.
   • Incorrect. Is er een hydrofiel of een hydrofoob membraan nodig om eiwitten te binden ?
3. Het hydrofobe membraan wordt door toevoegen van water geactiveerd. Hierdoor verkrijgt het membraan de volle eiwit binding capaciteit.
   • Incorrect. Wat gebeurt er als een hydrofoob membraan in aanraking komt met water?
4. Het hydrofobe membraan wordt door toevoegen van methanol geactiveerd. Hierdoor verkrijgt het membraan de volle eiwit binding capaciteit.
   • Juist! Het membraan is hydrofoob zodat eiwitten eraan kunnen binden. Als je een waterige oplossing gebruikt, krijgt het membraan niet de volle eiwitbinding capaciteit. Daarom moet je voor de activatie van het membraan methanol gebruiken.

Which is the correct way to assemble a transfer stack? Fill in the correct answer:

Negative Terminal (anode) – [a] – [b] – [c] – [d] – Positive Terminal (cathode)

Think about which item you use more than once. Which way does the current flow? And on which side of the membrane, gel or transfer stack is the protein?

Correct answer = Negative Terminal (anode) – [a=transfer stack] – [b=gel] – [c=PVDF membrane] – [d=transfer stack] – Positive Terminal (cathode)

• Correct answer
  • Correct! In this configuration the current will draw the proteins from the gel onto the PVDF membrane. With the anode as the most down layer and the cathode on top, the SDS covered, negatively charged proteins will migrate from the gel onto the membrane.
• Incorrect answer b and c swapped
  • Incorrect. In this configuration the current draws the proteins into the transfer stack, resulting in an empty membrane.
• Incorrect answer c and d swapped
  • Incorrect. This configuration will not work, as the gel and PVDF membrane need to be in close proximity/next to each other in order for the proteins to migrate from the gel onto the membrane.
• Incorrect answer a and b swapped
  • Incorrect. This configuration will not work. There will be no correct current created as the transfer stack is part of the system to guide the electricity and is also connected to the reservoir buffer.