Protein Quantification Flashcards

1
Q

What are the three steps of protein quantification?

A
  1. Separate/purify
  2. Identify
  3. Quantify
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2
Q

What are four types of chromatography?

A

HPLC, Affinity, Size exclusion, and Ion exchange

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3
Q

What are the three detectors for proteins?

A
  1. UV
  2. Fluorescence
  3. Refractive Index
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4
Q

Where is the main action of chromatography occuring?

A

On the column

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5
Q

What is a way that a chromatography column can separate materials?

A

hydrophilicity

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6
Q

Explain how materials will come out in a chromatography column based on their hydrophilicity.

A

More hydrophilic materials will come out of the column first because of their high affinity for the mobile phase. Then the more hydrophobic materials will come out later

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7
Q

Intensity of protein absorption signal is (disproportionate or proportionate to concentration.

A

proportionate

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8
Q

Retention time is affected by ______ and ____.

A

Retention time is affected by the column and solvent.

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9
Q

Explain functional groups and how they come out of the column.

A

The longer chains they have, the more hydrophobic, and the quicker they come out of the column

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10
Q

Color is ___ to concentration.

A

proportional

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11
Q

Explain how ELISA works?

A

A plate that has a lot of capture antibodies and your protein will attach to the antibody. then you add a secondary biotin antibody that will interact with your protein. Then add an enzyme and substrate that will produce a color. If you observe a color, you know your protein is there.

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12
Q

What is biotin streptevadin?

A

A large protein with dimers

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13
Q

What is biotin commonly used for?

A

conjugation

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14
Q

What is an advantage of ELISA?

A

It is very sensitive and you can detect very small amount of protein

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15
Q

What is important to know before you choose a quantification method?

A

An approximate concentration range because each method has an accuracy range

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16
Q

What are the advantages of chromatography?

A

easy, multiple columns and solvents available, very sensitive to small amounts of proteins

17
Q

What are disadvantages to chromatography?

A

Only be used for proteins in solution (cant use for proteins on the surface

18
Q

What are the advantages for colorimetric and fluorescent assays?

A

surface proteins/solution, easy to get quantitative data

19
Q

What are the disadvantages for colorimetric and fluorescent assays?

A

many require pre-labeling

20
Q

What are the advantages for ELISA?

A

very sensitive to small amounts (pg), can also use on the surface and solution

21
Q

What are the disadvantages for ELISA?

A

requires antibodies to interact with the protein target, expensive

22
Q

What are the advantages for the western blot?

A

Can be used to probe multiple proteins in the same sample

23
Q

What are the disadvantages for the western blot?

A

requires antibodies to protein target, proteins must be in solution, less sensitive than ELISA