Biochemistry Test 2 - Protein Separation

Front 1

Western Blot

Back 1

Detect small amounts of cytoplasmic protein

1. SDS-PAGE (gel electrophoresis) of protein sample

2. If we were to stain now, it would be a mess, so:
Run onto a membrane (get invisible mirro image)

3. Incubate membrane w/ blocking sol'n (milk); binds to everywhere where there are no proteins

4. 1⁰ Ab ⇨ can be poly or mono

5. 2⁰ Ab (binds to 1⁰) ⇨ must be polyclonal (signal amplification)

** Enzyme conjugated to 2⁰: provides substrate that generates measurable signal (light, color, fluoro.)

Front 2

When might we use antibodies?

Back 2

1. Affinity Chromatography
2. IHC
3. Localization (in cell culture)
4. Western Blot / Protein Quantificatio
5. Protein Identification

Front 3

Polyclonal Antibody

Back 3

Recognizes many epitopes of Antigen

Front 4

In isoelectric focusing gel electrophoresis:

Back 4

there is a pH gradient that parallels the electric fild gradient

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Procedure necessary to test whether amino acid sequence of ribonuclease contains all the information needed for it to fold into its native, fucnal 3-d structure

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1. FIRST: add 8M urea; RESULT: denatures, partially unfolds protein
2. FIRST: add reducing agent; RESULT: breaks S-S, RNase now completely unfolds
3. FIRST: dialyze away urea; RESULT: removal of denaturant, partial re-folding
4. FIRST: gendle air oxidation; RESULT: disulfides reform, final correct folding, yielding native form

Front 6

Salting Out (precipitation)

Back 6

Can be used to fractionate proteins since most are less soluble at high [salt]. This concentration (at which protein is no longer soluble) is diff for diff proteins
-> use [diff] depending on what you want to precipitate

Front 7

Dialysis

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Semipermeable membrane -> proteins too big to leave bag, while small molecules may leave
* useful to get rid of salt, although it won't sep diff proteins

Front 8

Gel Filtration Chromatography

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SIZE: LARGE=FAST, SMALL=SLOW
* gel (such as agarose) contains many beads, large molecules flow around them, small molecules get stuck in them

Front 9

Ion Exchange Chromatography

Back 9

CHARGE: (+) = SLOW, (-)=FAST
* gel contains neg charged beads (which contain carboxylate group); pos proteins will bind and take longer to run. (can also contain beads w/ (+) charge)

Front 10

Affinity Chromatography

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FUNCTIONAL GROUPS
* separat'n based on a highly specific biologic interaction such
-> antigen and antibody
-> enzyme and substrate
-> receptor and ligand.

Front 11

HPLC

Back 11

* similar to other column techniques but column materials are much finer
-> more interaction sites = greater resolving power
-> pressure added to obtain adeq. flow rate

Front 12

HIS-tags

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Attached to proteins of interest and run down gel containing immobilized nickel (II)

-> HIS-tag binds tightly to metal ions, binding desired protein while other proteins flow though the column

* protein eluted by addition of imidazole or another molecule that displaces it from resin

Front 13

Gel Electrophoresis

Back 13

velocity=Ez/f
f-depends on mas and shape and viscosity of medium)
z=charge
E=electric field strength

Front 14

Direction to run Gel Electrophoresis

Back 14

RUN TO RED
(-) --> (+)

Front 15

To separate based on mass only

Back 15

1. SDS (anionic detergent): denatures by disrupting noncovalent interactions
2. mercaptoEtOH: reduces disulfide bonds
3. Anion of SDS binds to main chain
* Complex now has large net (-) charge -> runs based on mass
* separate w/electrophoresis
* visualize w/coomasie blue
* if radioactively labeled, arg (x-ray film over gel)

Front 16

Isoelectric Focusing

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CONTENT OF ACIDIC & BASIC RESIDUES
* establish pH gradient & add voltage
* proteins stop moving at their isoelectric point (pH at which net charge is zero)

Front 17

Methods for cleaving polypeptides (and bond cleaved)

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1. CNBr: Met-X
2. Trypsin: Lys-X and Arg-X
3. Chymotrypsin: Hydrophobic AA-X
4. Hydroxylamine: Asn-Gly
5. Mild acid: Asp-Pro

Front 18

Mass Spectrometry

Back 18

DISCRIMINATES MOLECULES BASED ON MASS:
* sample in a vacuum is vaporized under protonating conditions, imparting a (+) charge

* electrical field propels the cations through a magnetic field which deflects & focuses them onto a detector

* the magnetic force required to deflect each ion is recorded (current applied to the electromagnet)

* for ions of identical net charge, this force is proportionate to their mass

Front 19

What can be used to activate the carboxyl group?

Back 19

dicyclohexylcarbodiimide (DCC)

Front 20

What can be used to protect the amino group?

Back 20

t-Boc

Front 21

Reagent used to determine amino acid sequence of a small peptide

Back 21

phenyl isothiocyanate

Front 22

Reagent used in reversable denaturation of a protein without any disulfide bonds

Back 22

urea

Front 23

Reagent used in hydrolysis of peptide bonds on the carboxyl side of aromatic residue

Back 23

chymotrypsin

Front 24

Reagent used in the cleavage of peptide bonds on the carboxyl side of methionine

Back 24

CNBr

Front 25

Reagent used in hydrolysis of peptide bonds on the arboxyl side of lysine and arginine

Back 25

trypsin

Front 26

Reagents (2) used in reversible denaturation of a protein that contains disulfide bonds

Back 26

urea, B-mercaptoethanol

Front 27

Reagent needed to activate carboxyl groups during peptide synthesis

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dicyclohexylcarbodiimide (DCC)

Front 28

Reagent needed to determine the amino acid composition of a small peptide

Back 28

6N HCl, cation exchanger

Front 29

Reagent needed to remove t-Boc protecting group during peptide synthesis

Back 29

dilue F3CCOOH

Front 30

What reagent is used in Edman degredation? (for sequencing small amino acids)

Back 30

Phenylisothiocyanate

Ph-N=C=S

Front 31

What bond does CNBr cleave?

Back 31

Met-X

Front 32

What bonds does Trypsin cleave?

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Lys-X and Arg-X

Front 33

What bonds does Chymotrypsin cleave?

Back 33

hydrophobic amino acid - X

Front 34

What would you use to cleave Lys-X bond?

Back 34

Endoprotease Lys-C

Front 35

What would you use to cleave Arg-X bond?

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Endoprotease Arg-C

Front 36

What would you use to cleave X-Asp?

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Endoprotease Asp-N

Front 37

What bond does endoprotease Asp-N cleave

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X-Asp

Front 38

What would you use to cleave Asn-Gly

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Hydroxylamin

Front 39

What would you use to cleave Trp-X bond?

Back 39

o-lodosobenzene

Front 40

What is Mass Spec used for?

Back 40

Deteriming the mass of small proteins. Also useful for detecting the phosphate, hydroxyl, and other groups o posttranslational modified amino acids.

Front 41

Large organic compounds are difficult to volatilize, making it hard to do Mass Spec on them. What's one way to overcome this?

Back 41

MALDI = matrix assisted laser-desorption.

* peptides are eluted from a reversed phase HPLC column - introduced directly into mass spec for immediate determination of mass.

Front 42

Antigen

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Foreign to substance to which antibody binds; given to animal to elicit production of antibody

Front 43

Antibody

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Immunoglobin = proetin synthesized by animal in response to antigen (foreign substance). Binding is a step in immune response.

Front 44

Antigenic determinant

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= Epitope = The specific group of cluster of amino acids on target antigent (target molecule) that antibody binds to

Front 45

What technique can be used to localize specific protein in an intact cell

Back 45

Immunoelectron microscope (better view) & fluroscent microscope

Front 46

What is a commonly used protecting group in solid phase protein synthesis?

Back 46

t-Boc

Front 47

In X-ray crystallography, what is the protein resolution limit?

Back 47

2A

Front 48

How can we use X-Ray crystallography to look at enzyme-substrate linkage?

Back 48

If an enzyme is crytallized with and without its subtrate, the different in 3D structure should help determine how enzyme binds

Front 49

What are the 3 major classes of RNA found in E. coli?

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1. rRNA (one per ribosome; catalyst for protein synth)

2. mRNA (template for protein synthesis = translation)

3. tRNA (caries AAs in sequence determined by mRNA & in activated form to ribosomes for peptide bond formation)

Front 50

More specifically, what is tRNA?

Back 50

Transfer RNA is the "adapter molecule" which carries activated amino acids to the template.

* contain an amino acid attachment site
* contain a template recognition site = anticodon

Front 51

Codon

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the sequence on mRNA to which tRNA recognizes

Front 52

Anticodon

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the template recognition sequence on tRNA, complementary to mRNA codon

Front 53

Four points of the genetic code

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1. three nucleotides encod AA

2. code is nonoverlapping

3. code has no punctuation

4. code is degenerate (more than one code per aa)

Front 54

In genetics, what is a synonym?

Back 54

Triplets that encode the same amino acid; usually differ only in the last base

Front 55

Southern Blotting

Back 55

Separtes DNA:
1. Digest DNA w/ restriction enzymes
2. Run samples on an agarose gel
3. Denature (separate double strands) of DNA by soaking gel in 0.5M NaOh
4. Transfer ssDNA onto nitrocellulase membrane
5. Treat membrane with UV light to covalently bond ssDNA to membrane
6. Incubate with radiolabeled ssDNA probe (ex: 32P ATP) -> ssDNA and probe should hybridize
7. Visualize (ARG)

Front 56

Bragg's Law

Back 56

Tells us about spacing of crystals in a lattice (in X-ray crystallography)

Front 57

(3) Physical principles underlying x-ray crystallography

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1. electrons scatter x-rays

2. scattered waves recombine (inhance or cancel each other if they are in phase or out)

3. the way in which electrons scatter x-rays depends on atomic arrangment

Front 58

Fourier Transformation

Back 58

In X-Ray crystallography = mathematical method for constructing image out of x-ray data
* for each spot, gives wave showing e- density: amplitude is proportional to square root of observed intensity

*each wave has a phase - determines whether others are in phase our out (inhances or cancels)

* gives 3D e- distribution

Front 59

How to heavy atoms scatter X-rays compared to light atoms?

Back 59

Heavy atoms (many e-) scatter more effectively

Front 60

What is X-ray crystallography used for?

Back 60

Determining 3D structure of protein

Front 61

What happens when an electric field is applied in NMR?

Back 61

Proton can take on one of two spin states:
a - lower energy
b - higher energy

Add energy to get a-spin state to b-spin state: transformation gives us NMR line.

Front 62

In NMR, how do you get a resonance spectrum?

Back 62

Keep magnetic field constant, vary electromagnetic radiation: this moves protons from a-spin state to b-spin state; transition gives us NMR line

Front 63

What does DNA ligase do?

Back 63

Joins together fragments of DNA
- okazaki fragments, or in cloning

Front 64

What is cytogenetic mapping?

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Anchors genes or markers on to chromosomes
* carried out by flurosence in in situe hybridization (FISH)
* similar to southern blotting: probe with gene of interest is used to hybridize to chromosome containing complementary sequence

Front 65

Paralog

Back 65

Type of homolog (derived from common ancestor):
Present within one species, usually differ in function

Front 66

Ortholog

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Type of homolog (derived from common ancester):
Present within two different species, but have similar functions

Front 67

Distal Histidine i Hb

Back 67

Bound to the top of Fe - it is what oxygen binds to