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Flashcards in Protein Diagnostic Methods Deck (28):
1

What are the two methods that obtain the 3-D structure of a protein with high resolution?

1) X-ray crystallography
2) NMR

2

What are the two methods that obtain the 3-D structure of a protein with low resolution?

1) Circular Dichroism (CD)
2) Electron tomography

3

X-Ray crystallography (Steps, pros, cons)

1) Steps: (purify protein, crystallize protein, collect diffraction data, calculate electron density, fit residues into density) 2) Pros: (No size limits, well established) 3) Cons: (Difficult for membrane proteins, cannot see hydrogens, relevance to soluble structure is unclear)

4

NMR (Steps, Pros, Cons)

1) Steps: purify the protein, dissolve the protein, collect NMR data, assign NMR signals, calculate structure. 2) Pros: no need to crystallize the protein, can see many hydrogens, can show which regions are flexible. 3) Cons: difficult for insoluble proteins, flexible proteins, works best with small proteins.

5

Electron Tomography (Steps, Pros, Cons)

1) Steps: purify the protein, freeze the protein, collect electron density tomograph data, average the images to get high-reliability volumes, calculate the structure. 2) Pros: no need to crystallize the protein, low density regions are frequently lost 3) Cons: freezing proteins causes complications, works best with large proteins with known subunit crystal structures.

6

Homology Modeling

Does not determine the true structure. Generates a structural hypothesis. Useful due to the rise in the amount of genomes sequenced. Takes a little time.

7

Why do high resolution studies (2)

1) Exceptionally high quality structure information
2) Can greatly increase the understanding of a protein's function

8

Why not do high-resolution studies? (3)

1) Potentially time consuming
2) Technically challenging (you need a specialist)
3) enzymatic function does not require a structure to answer effectively.

9

Why do a low-resolution study? (2)

1) Confirm that your protein of interest still has structure (after manipulation in the lab) 2) Significantly faster.

10

Why do a modeling study?

1) Because you need structural data, and there are high-similarity family members available and you lack the time or expertise to do a high-resolution study, or are working with a difficult protein.

11

What are the five steps to get a purified protein?

1) Make a crude extract 2) separate the protein components (separate based on charge, size, solubility, thermal stability, affinity for a ligand, affinity for a ligand, hydrophobicity 3) Determine the purity of the protein. 4) Measure the total amount of protein 5) Calculate purification factor and yield.

12

How do you separate a protein on the basis of charge?

1) Ion-exchange chromatography 2) Exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH

13

How do you separate proteins on the basis of size?

1) Size exclusion chromatography. 2) protein mixture is added to column containing cross-linked polymer, proteins separate by size with larger molecules passing more freely

14

How do you separate proteins on the basis of affinity?

1) Affinity Chromatography 2) Solution of ligand is added to column, protein mixture is added to the column. The protein that binds the ligand does not elute, while others do.

15

What are the two methods used to determine the purity of a protein?

1) PAGE 2) SDS PAGE

16

What are the disadvantages of using PAGE?

PolyAcrylamide Gel Electrophoresis uses an electric field to pull proteins according to their charge. Separation becomes based on size:charge:shape ratio, which is complicated.

17

What is the advantage of using SDS PAGE?

Sodium Dodecyl Sulfate is used to coat proteins and give them an even charge:mass ratio, so they are ultimately size distributed.

18

Isoelectric focusing

Separates proteins according to their isoelectric points. Proteins are applied to a gel with differing levels of pH. An electric field is applied, the proteins migrate to the pH equivalent to its pI.

19

What are the two methods that can be used to measure the total amount of proteins?

1) Absorbance at 280 nm- Aromatic side chains (tyrosine & tryptophan) absorb UV light
2) Pierce Assay (modified Bradford assay) positvely charged amino acids bind to a dye (Coomassie G-250 and once bound, absorb light at 595 nm)

20

Absorbance at 280 (Method, Pros, Cons)

1) Used to measure the amount of protein in a sample. Absorbance at 280 nm- Aromatic side chains (tyrosine & tryptophan) absorb UV light. 2) Pros: fast, easy, quantitative/ extremely accurate if a single protein of high purity is measured, since the ratio of Tyr/Trp to mass is known. 3) Cons: Not all proteins have equal amounts of Tyr/Trp. Thus very inaccurate for bulk proteins/Non-protein compounds absorb at 280 nm

21

Pierce/Bradford Assay (Method, Pros, Cons)

1) Used to measure the total amount of protein. positvely charged amino acids bind to a dye, Coomassie G-250, and once bound, absorb light at 595 nm. 2) Pros: Most proteins bind to Coomassie-G-250 evenly thus high accuracy for bulk proteins. Few non protein compounds absorbs at 595 nm. 3) Cons: Takes longer than simply measuring absorbance, and must measure standards and replicate samples for accuracy.

22

Purity calculation

Purity = Amount of the protein of interest/Amount of total protein

23

Yield calculation

Yield = final amount of protein/initial amount of protein

24

What two methods are used to identify unknown proteins?

MALDI MS and ESI MS

25

What are the advantages/disadvantages of Mass Spec?

Advantages: Can be used w/ small amount of proteins and mixed samples. Provides sequence information Disadvantages: fragmentation processes leave gaps in the sequences

26

Circular Dichromism (CD)

Measurements of the differences in absorption of left handed and right handed polarized light

27

Cation-exchange chromatography

Solid negative matrixl in mobile phase polar proteins move more slowly

28

Edman Degradation

Process that determines the amino acid sequence of a protein by removing individual AAs

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