• After imaging Cy-5, the assistant will carefully remove the foil and cover the blots near the antibodies with ECL to image the chemiluminescence signal.
• For this, Reagent A and Reagent B are pipetted in a 1: 1 ratio into a 15 ml tube (250 µl per blot).
• The blot is covered again with cling film (watch out for air bubbles!).

Welk antilichaam detecteer je nu?

a.      Rabbit-Alexa 647
Correct = yes

b.     Mouse-Alexa 647
Correct = yes

c.      Rabbit-HRP
Correct = no

d.     Mouse-HRP
Correct = no

Feedback if incorrect: Niet goed. Kijk nog een keer naar de datasheets van de antilichamen. Deze zijn te vinden in het materialenoverzicht.

Welk antilichaam detecteer je nu?

a.      Rabbit-Alexa 647
Correct = no

b.     Mouse-Alexa 647
Correct = no

c.      Rabbit-HRP
Correct = yes

d.     Mouse-HRP
Correct = yes

Feedback if incorrect: Niet goed. Kijk nog een keer naar de datasheets van de antilichamen. Deze zijn te vinden in het materialenoverzicht.

Which antibodies will be imaged in which channel?

1. IgG-HRP is imaged through Cy5 because it contains a label which fluoresces at 630 nm indicating the presence of anti-α-Tubulin and Alexa Fluor 647 is imaged through Direct imaging because the substrate present in ECL prime solution is catalysed by HRP resulting in luminescence indicating the presence of anti-p-ERK and anti-p-AKT
   • Incorrect. Think back to which primary antibody is linked to which secondary antibody.
2. Alexa Fluor 647 is imaged through Cy5 because it contains a label which fluoresces at 630 nm Indicating the presence of anti-p-ERK and anti-p-AKT and IgG-HRP is imaged through Direct imaging because the substrate present in ECL prime solution is catalysed by HRP resulting in luminescence Indicating the presence of anti-α-Tubulin
   • Incorrect. Think back to which primary antibody is linked to which secondary antibody.
3. IgG-HRP is imaged through Cy5 because it contains a label which fluoresces at 630 nm Indicating the presence of anti-p-ERK and anti-p-AKT and Alexa Fluor 647 is imaged through Direct imaging because the substrate present in ECL prime solution is catalysed by HRP resulting in luminescence Indicating the presence of anti-α-Tubulin
   • Incorrect. Think back to which primary antibody is linked to which secondary antibody.
4. Alexa Fluor 647 is imaged through Cy5 because it contains a label which fluoresces at 630 nm Indicating the presence of anti-α-Tubulin and IgG-HRP is imaged through Direct imaging because the substrate present in ECL prime solution is catalysed by HRP resulting in luminescence Indicating the presence of anti-p-ERK and anti-p-AKT
   • Correct! As answered in a previous question, the secondary antibody IgG-HRP is used for the detection of both Anti-p-Akt and anti-p-ERK, while Alexa Fluor is used for the detection of anti-α-Tubulin. Alexa Fluor 647 is detected through fluorescence, while HRP is dependent on a chemical reaction.

Which statement about excitation and emission wavelengths is correct?

1. Every fluorophore has one specific excitation and emission wavelength.
   • Not correct, there is not one specific wavelength to excite a fluorophore. Neither is there one specific wavelength emitted after excitation of the fluorophore.
2. Every fluorophore has a spectrum of excitation and emmision wavelengths, and we choose a most optimal wavelength for imaging.
   • Correct!
3. Every fluorophore has 1 specific excitation wavelenght and a spectrum of emmission wavelengths, which we optimize to retrieve a maximal signal.
   • Not correct, there is not one specific wavelength to excite a fluorophore.

Why is it sometimes necessary to use a secondary antibody (indirect detection)?

1. Using only the first antibody is not reliable.
   Correct = no
2. The detection signal will be amplified.
   Correct = no
3. The first antibody is not detectable.
   Correct = yes

Feedback if correct: Correct! Multiple secondary antibodies can bind on one first antibody. This way, more substrate can be converted by the bound enzyme. The detection signal will then be amplified. The use of the first and second antibody combination is also called indirect detection.

Feedback if incorrect: Why would you choose indirect detection?

On which mechanism is chemiluminescence based on?

1. A secondary antibody is conjugated to an enzyme such as horseradish peroxidase (HRP). When the secondary antibody binds with the first antibody, an immune reaction will start what emits light.
   Correct = no
2. A first or secondary antibody is conjugated to an enzyme such as horseradish peroxidase (HRP). When substrate molecules are added, a reaction will start, catalysed by the bound enzymes. This reaction emits light.
   Correct = no
3. A first or secondary antibody is conjugated to an enzyme such as horseradish peroxidase (HRP). When enzymes are added a reaction starts, catalysed by the enzyme. This reaction emits light.
   Correct = yes

Feedback if correct: Correct! This reaction emits light.

Feedback if incorrect: How is light initiated?