Biotech-immunoprecipitation Flashcards Preview

Biotechnology > Biotech-immunoprecipitation > Flashcards

Flashcards in Biotech-immunoprecipitation Deck (23)
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1
Q

What is used to re-suspend the protein A slurry?

A

lysis buffer

2
Q

What is pre-clearing?

A

Preclearing the lysate at the start of each immunoprecipitation experiment is a way to remove potentially reactive components from the cell lysate prior to the IP to prevent the non-specific binding of these components to the IP beads or antibody.

3
Q

what is done to the precleared lysate once it is added to tubes?

A

parafilm to prevent sample loss.

4
Q

At what temperature and for how long are the samples to be IP’d incubated for?

A

90 minutes at 4C

5
Q

How are the immuno-precipitated complexes collected after the IP?

A

Centrifugation at 5000 rpm for 1 minute at room temperature. Take supernatant and leave pellet (which should contain the important stuff)

6
Q

After IP, and after centrifugation, what does the supernatant contain?

A

Most likely proteins with non-specific interactions or cell debris.

7
Q

What does the pellet contain?

A

hopefully the protein of interest bound to the antibody and bound to the protein a beads.

8
Q

How are the pellets from IP resuspended?

A

Using laemmli sample buffer contain beta-mercaptoethanol.

9
Q

What does beta-mercaptoethanol do?

A

reduce disulphide bonds (important for SDS, obviously)

10
Q

What samples are resuspended?

A

Those before IP and those after IP

11
Q

After the samples have been resuspended, what else is done?

A

Heat all samples at 95C for 5 minutes, vortex and spin for 2 minutes.

12
Q

What is the maximum voltage limit?

A

100 VDC

13
Q

What is the max power limit?

A

40 Watts

14
Q

What is the maximum ambient temperature limit?

A

50C

15
Q

Why are we using chilled (4C) transfer buffer?

A

improves heat dissipation

16
Q

how is the PVDF membrane activated?

A

By soaking in methanol for 30 seconds

17
Q

Once the membrane is activated, where is it added?

A

transfer buffer

18
Q

How long is the blot run for and what conditions?

A

100V for 1 hour

19
Q

What time interval should the electroblot be monitored%

A

every 15 minutes

20
Q

What should the readings be (what should they be adjusted to every 15 minutes)?

A

reduce or increase voltage to keep current at around 300-350 mA.

21
Q

How is the membrane removed?

A

using tweezers and wrap in saran wrap

22
Q

How are most things cleaned?

A

with deionized water

23
Q

what is used to refill the bio-ice cooling unit?

A

distilled water

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