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67 Cards in this Set

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Western Blot
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.)
When might we use antibodies?
1. Affinity Chromatography
2. IHC
3. Localization (in cell culture)
4. Western Blot / Protein Quantificatio
5. Protein Identification
Polyclonal Antibody
Recognizes many epitopes of Antigen
In isoelectric focusing gel electrophoresis:
there is a pH gradient that parallels the electric fild gradient
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
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
Salting Out (precipitation)
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
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
Gel Filtration Chromatography
* gel (such as agarose) contains many beads, large molecules flow around them, small molecules get stuck in them
Ion Exchange Chromatography
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)
Affinity Chromatography
* separat'n based on a highly specific biologic interaction such
-> antigen and antibody
-> enzyme and substrate
-> receptor and ligand.
* similar to other column techniques but column materials are much finer
-> more interaction sites = greater resolving power
-> pressure added to obtain adeq. flow rate
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
Gel Electrophoresis
f-depends on mas and shape and viscosity of medium)
E=electric field strength
Direction to run Gel Electrophoresis
(-) --> (+)
To separate based on mass only
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)
Isoelectric Focusing
* establish pH gradient & add voltage
* proteins stop moving at their isoelectric point (pH at which net charge is zero)
Methods for cleaving polypeptides (and bond cleaved)
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
Mass Spectrometry
* 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
What can be used to activate the carboxyl group?
dicyclohexylcarbodiimide (DCC)
What can be used to protect the amino group?
Reagent used to determine amino acid sequence of a small peptide
phenyl isothiocyanate
Reagent used in reversable denaturation of a protein without any disulfide bonds
Reagent used in hydrolysis of peptide bonds on the carboxyl side of aromatic residue
Reagent used in the cleavage of peptide bonds on the carboxyl side of methionine
Reagent used in hydrolysis of peptide bonds on the arboxyl side of lysine and arginine
Reagents (2) used in reversible denaturation of a protein that contains disulfide bonds
urea, B-mercaptoethanol
Reagent needed to activate carboxyl groups during peptide synthesis
dicyclohexylcarbodiimide (DCC)
Reagent needed to determine the amino acid composition of a small peptide
6N HCl, cation exchanger
Reagent needed to remove t-Boc protecting group during peptide synthesis
dilue F3CCOOH
What reagent is used in Edman degredation? (for sequencing small amino acids)

What bond does CNBr cleave?
What bonds does Trypsin cleave?
Lys-X and Arg-X
What bonds does Chymotrypsin cleave?
hydrophobic amino acid - X
What would you use to cleave Lys-X bond?
Endoprotease Lys-C
What would you use to cleave Arg-X bond?
Endoprotease Arg-C
What would you use to cleave X-Asp?
Endoprotease Asp-N
What bond does endoprotease Asp-N cleave
What would you use to cleave Asn-Gly
What would you use to cleave Trp-X bond?
What is Mass Spec used for?
Deteriming the mass of small proteins. Also useful for detecting the phosphate, hydroxyl, and other groups o posttranslational modified amino acids.
Large organic compounds are difficult to volatilize, making it hard to do Mass Spec on them. What's one way to overcome this?
MALDI = matrix assisted laser-desorption.

* peptides are eluted from a reversed phase HPLC column - introduced directly into mass spec for immediate determination of mass.
Foreign to substance to which antibody binds; given to animal to elicit production of antibody
Immunoglobin = proetin synthesized by animal in response to antigen (foreign substance). Binding is a step in immune response.
Antigenic determinant
= Epitope = The specific group of cluster of amino acids on target antigent (target molecule) that antibody binds to
What technique can be used to localize specific protein in an intact cell
Immunoelectron microscope (better view) & fluroscent microscope
What is a commonly used protecting group in solid phase protein synthesis?
In X-ray crystallography, what is the protein resolution limit?
How can we use X-Ray crystallography to look at enzyme-substrate linkage?
If an enzyme is crytallized with and without its subtrate, the different in 3D structure should help determine how enzyme binds
What are the 3 major classes of RNA found in E. coli?
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)
More specifically, what is tRNA?
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
the sequence on mRNA to which tRNA recognizes
the template recognition sequence on tRNA, complementary to mRNA codon
Four points of the genetic code
1. three nucleotides encod AA

2. code is nonoverlapping

3. code has no punctuation

4. code is degenerate (more than one code per aa)
In genetics, what is a synonym?
Triplets that encode the same amino acid; usually differ only in the last base
Southern Blotting
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)
Bragg's Law
Tells us about spacing of crystals in a lattice (in X-ray crystallography)
(3) Physical principles underlying x-ray crystallography
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
Fourier Transformation
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
How to heavy atoms scatter X-rays compared to light atoms?
Heavy atoms (many e-) scatter more effectively
What is X-ray crystallography used for?
Determining 3D structure of protein
What happens when an electric field is applied in NMR?
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.
In NMR, how do you get a resonance spectrum?
Keep magnetic field constant, vary electromagnetic radiation: this moves protons from a-spin state to b-spin state; transition gives us NMR line
What does DNA ligase do?
Joins together fragments of DNA
- okazaki fragments, or in cloning
What is cytogenetic mapping?
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
Type of homolog (derived from common ancestor):
Present within one species, usually differ in function
Type of homolog (derived from common ancester):
Present within two different species, but have similar functions
Distal Histidine i Hb
Bound to the top of Fe - it is what oxygen binds to