• Take your tubes from the roller bench and remove blocking solution from the tubes.
• Gently rinse your membrane 1x with 10 ml TBS-T.
• Carefully pour approx. 10 ml of TBS-T into the tube, screw the cap and gently invert the tube 3x.
• Remove  TBS-T from the tube.
• ·         Wash the membrane for 5 minutes with 5 ml TBS-T on the roller bench.

• Remove TBS-T from the tubes again.
• You will receive pAkt- and pErk antibodies + anti-tubulin diluted in TBS-T/1% BSA from the assistants.
• If the calculated amount is not correct, you have to recalculate it before you get your antibodies.
• Add 3 ml of the adequate primary antibody to the tubes containing the membranes.

Incubate overnight on the roller bench at 4 °C for pERK. Incubate overnight at RT for pAkt.

Label the tubes properly! So you can find back your tubes tomorrow and that you still know which antibody is in which tube!

Take your tubes from the roller bench (4^oC) and remove the 1^st antibody solution from the tubes by pouring it in a new 50 ml tube. p-AKT and p-ERK separate!

The 1^st antibodies are collected and stored for reuse.

• Gently rinse your membrane 1x with 10 ml TBS-T.
• Carefully pour approx. 10 ml of TBS-T into the tube, screw the cap and gently invert the tube 3x.
• Remove  TBS-T from the tube.
• Wash the membrane 3x 5 min with 10 ml TBS-T on the roller bench (RT)

• For each membrane you need secondairy antibodies in 3ml of TBS-T/1% BSA
• Calculate how much of each antibody is needed and show your calculations to the assistant.
• The assistants will add the secondary antibodies.
• If the calculated amount is not correct, you have to recalculate it before you get your antibodies.
• Wrap the antibody tubes with aluminum foil
• Remove TBS-T from the tube.
• Add your secondary antibodies to the tubes with your membrane, 3 ml each.
• Is your tube still clearly labeled? So you can find back your tubes and that you still know which antibody is in which tube!

Incubate for 1 hour at room temperature on the roller bench.

• Take your tubes from the roller bench and remove the 2^nd antibody solution from the tubes by pouring it in a new 50 ml tube.
• The 2nd antibodies are collected and stored for reuse.
• Gently rinse your membrane 1x with 10 ml TBS-T.
• Carefully pour approx. 10 ml of TBS-T into the tube, screw the cap and gently invert the tube 3x.
• Remove  TBS-T from the tube.
• Wash the membrane for 5 minutes  with 5 ml TBS-T on the roller bench.
• After the last wash step, leave your membrane in the TBS-T: your membrane must not dry out !!!

Indicate which label is conjugated to which antibody or antibodies. You can check the antibody information for the western blot in the datasheet provided for this course.

Why is it important to know which animal produced the antibody?

1. Because the first and second antibody should be from the same animal.
   • Incorrect. How does an antibody work?
2. To research if it will create an immune response in the cells.
   • Incorrect. What do you want to accomplish with the antibodies?
3. To be sure that the secondary antibody targets the first antibody.
   • Correct! The first and secondary antibody should be produced by different animals. Antibodies bind to foreign substances.

What is the correct course of action after adding the 2nd antibody to the membrane?

1. Cover with aluminium foil and place on the roller bank
   • Correct! The tubes must be placed on the roller bank for proper coverage with the second antibody, and the tube must be covered in foil because Alexa Fluor-647 is light-sensitive and will degrade with exposure to light.
2. Cover the tube in aluminium foil
   • Partly correct. The secondary antibodies are light sensitive, so we need to incubate in the dark. But do we have a proper incubation?
3. Place the tube on the roller bank
   • Partly correct. We need movement for a proper incubation. But also remember the function of the secondary antibodies.
4. Cover with aluminium foil and place on the roller bank at 4C.
   • Incorrect! What is the funtion of incubation at 4C?

What is the purpose and result of washing the membrane between adding the 1st and 2nd antibodies?

1. The purpose is to wash out any remaining primary antibodies so that the secondary antibodies do not attach to unbound primary antibodies, but only to ones attached to the target proteins.
   • Correct, but incomplete answer.
2. The purpose is to wash out any left-over milk protein residue left over, because this may interfere with the binding of the secondary antibody.
   • Correct, but incomplete answer.
3. The purpose is to wash the primary antibody off the target proteins in order for the secondary antibodies to be able to attach correctly.
   • Incorrect.
4. Both answers 1 and 2 are correct.
   • Correct!

In order to detect the protein of interest, both primary and secondary antibodies are used in Western Blotting. The previous question already showed which antibodies will be used in this experiment.

The dilution factor of the primary antibody solutions are:

·       rabbit-anti-p-Akt (1:2000)

·       rabbit-anti-p-ERK (1:1000)

·       mouse-anti-α-Tubulin (1:1500).

We need 3 ml diluted AB in 1% BSA in TBS-T.

What amount (in µL) of the primary antibodies is needed for the primary antibody staining?

1. 3 µL rabbit-anti-p-Akt; 2 µL rabbit-anti-p-ERK and 1.5 µL mouse-anti-α-Tubulin.
   • Incorrect. Did you use the correct dilution factor?
2. 1,5 µL rabbit-anti-p-Akt; 3 µL rabbit-anti-p-ERK and 2 µL mouse-anti-α-Tubulin.
   • Correct! The working solution is 3 mL 1% BSA in TBS-T. The primary antibody solutions are: rabbit-anti-p-Akt (1:2000), rabbit-anti-p-ERK (1:1000) and mouse-anti-α-Tubulin (1:1500). Using a dilution factor in the calculation, we divide the two concentrations and use 1/dilution factor x end volume formula. The dilution factors are 1/2000, 1/1000, 1/1500 and the formula will give 1/2000 x 3=1,5 µL, 1/1000 x 3= 3 µL and 1/1500 x 3=2 µL respectively.
3. 1,5 µL rabbit-anti-p-Akt; 1 µL rabbit-anti-p-ERK and 0,75 µL mouse-anti-α-Tubulin.
   • Incorrect. Did you use the correct dilution factor?
4. 0,75 µL rabbit-anti-p-Akt; 1,5 µL rabbit-anti-p-ERK and 1 µL mouse-anti-α-Tubulin.
   • Incorrect. Did you use the correct dilution factor?

To visualize the protein of interest, we need secondary antibodies to complete our complex.

The dilution factor of the secondary antibody solutions are:

·       anti-rabbit IgG-HRP (1:2000)

·       anti-mouse-Alexa Fluor 647 (1:2000)

We need 3 ml diluted AB in 1% BSA in TBS-T.

What amount of the secondary antibodies is needed?

We need [a] μl of anti-rabbit igG-HRP, then [b] µL of anti-mouse-Alexa Fluor 647.

• What amount of 1% BSA in TBS-T do we need?
• Check the dilution factor again.
• The dilution factor is the same for both secondary antibodies.

Correct answer: a = 1.5 and b = 1.5

Options

·       0.75

·       1

·       1.5

·       2

·       3

Correct answer:
Very good! The working solution is 3 mL 1% BSA in TBS-T. The secondary antibody solutions are: anti-rabbit IgG-HRP (1:2000) and anti-mouse-Alexa Fluor 647 (1:2000). Using a dilution factor in the calculation, we divide the two concentrations and use 1/dilution factor x end volume formula. The dilution factor is 1/2000 and the formula will give 1/2000 x 3=1,5 µL for both secondary antibody solutions.