Biotech-western blotting and chemiluminescence Flashcards

1
Q

Once the membrane is taken out of the fridge, it must be reactivated. How is this done?

A

Brief (30 second) incubation period in methanol.

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2
Q

How is the methanol rinsed off?

A

By incubating the activated membrane in water.

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3
Q

What is the blocking solution?

A

TBST buffer with 5% skim milk.

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4
Q

What is the point of the blocking solution?

A

Blocking solution works by covering the unoccupied areas of the membrane with a dense layer of molecules. This prevents non-specific binding of primary and secondary antibodies.

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5
Q

Once the blocking buffer is decanted, what is added?

A

The primary antibody solution.

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6
Q

Once the blocking solution is added, how long is this incubated for?

A

1 hour on orbital shaker at room temperature

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7
Q

What is the primary antibody solution?

A

1:2000 dilution of rabbit anti-PEN antibody in 1% skim milk in 1x TBST

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8
Q

How long is the membrane incubated with the primary antibody solution?

A

For 1 hour with gentle agitation at room temperature

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9
Q

How is the blot that had been incubated with the primary antibody washed?

A

Washed by decanting the primary antibody solution and adding 30 mL of TBST to the membrane. This is washed for a period of 5 minutes with gentle agitation at room temperature. The wash solution is then decanted and this is repeated 2x.

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10
Q

What is the secondary antibody conjugate solution composed of?

A

1:5000 dilution of goat anti-rabbit conjugated HRP in 1% skim milk in 1x TBST.

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11
Q

How long is the blot and secondary antibody solution incubated for?

A

45 minutes using gentle agitation at room temperature.

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12
Q

How is the membrane washed?

A

decant and add TBST to the membrane, wash and agitate for 5 mins at RT. Repeat twice with additional TBST.

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13
Q

What is done to allow the blot to develop? (after the washing step post-secondary antibody solution)

A

Remove the membrane and drain the excess liquid without letting the blot dry. Place on a piece of saran wrap on a level surface.

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14
Q

What is added to the 3mL screw cap containing the oxidizing agents?

A

Enhanced luminol reagent.

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15
Q

What is the ratio of oxidizing agent to luminol?

A

1:1, 1 mL of each

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16
Q

How is the oxidizing agent and luminol reagent mixed?

A

Vortex

17
Q

Immediately after vortexing, what is done with the solution?

A

Pipette the substrate solution evenly onto the membrane.

18
Q

How long is the substrate solution incubated with the membrane for?

A

1 minute

19
Q

How is the membrane removed?

A

Using tweezers, and draining the excess liquid from the blot.

20
Q

How is the reaction documented?

A

Using the Bio imaging system.