Analytic Techniques

Question 1

Technique in which antibodies are applied to proteins and separated via electrophoresis

Answer 1

Western blotting

Question 2

Technique that assesses the concentration of protein by measuring the extent to which the unlabeled proteins in the sample compete with radioactively labeled antigens for antibody binding sites

Answer 2

Radioimmunoassay

Question 3

Technique in which antibodies bind antigens, and amount of bound antibody is determined by reactions that produce visible signal, like color or fluroescence

Answer 3

ELISA

Question 4

Technique that quantifies amount of protein in a sample based on its absorption of light at a characteristic wavelength

Answer 4

Spectroscopy

Question 5

Region after centrifugation that would have protein of interest

Answer 5

Supernatant and would be above pellet

Question 6

How is protein of interest extracted?

Answer 6

Add protease inhibitors to protect from degrading elements of cell

Cool temp to protect from desaturation and minimize enzyme activity

Buffer: maintain pH of solution

Question 7

Salting in vs salting out:

Answer 7

Just below salt concentration that protein of interest is precipitated out of solution that can be filtered out

Question 8

protein purification

Answer 8

Extraction (protease inhibitors) > centrifugation (precipitate out of solution) > chromatography (depending on molecule of interest)

Question 9

Preparative vs Analytical purification

Answer 9

Preparative: large amount of desired protein

Analytical: small, relatively pure

Question 10

Separates proteins with different charges, relative number of acidic and basic residues

Answer 10

Isoelectric Focusing (IEF)
- molecule migrates until it reaches region of gel where pH is equal to pI

Question 11

Combines gel electrophoresis and IEF:

Answer 11

2D gel electrophoresis

Question 12

with regard to NMR, where is “downfield”?

Answer 12

Left handed side of spectrum, downshield = de shield

Question 13

Where is upfield in NMR?

Answer 13

Upfield is to the right, upfield is to be shield

Question 14

Where are downfield and upfield of NMR?

Answer 14

Deshield > destiny’s shieldren > Beyoncé to the left = downfield

Upfield = UP was the right movie

Question 15

in NMR de shielded refers to what phenomena?

Answer 15

Nuclei are near electronegative atoms

Question 16

Decanting acid in NMR, proton’s peak farthest downfield?

Answer 16

Downfield > deshield > destiny’s shieldren > Beyoncé to the left

• Farthest downfield will be the COOH of this molecule, carbon 1
• shift large distance, COOH proton is near two electronegative oxygen atoms
• COOH usually between 10 and 13 ppm

Question 17

Where is COOH signal usually appear on NRM spectrum and where?

Answer 17

Downfield, deshield, left, between 10 and 13 ppm

Question 18

Whats does mass spec peak represent?

Answer 18

Represents mass/charge ratio > fragmentation and ionization of molecule in question

Question 19

Are molecules ionized in mass spec or before the apparatus?

Answer 19

Done during mass spec not before

Question 20

How does ionization happen in mass spec?

Answer 20

Usually removal of one electron, leaving mass fragment unchanged in weight but differ in charge

Question 21

Several researchers test out a new mass spectrometry apparatus using a known sample of 3-pentanone. They note several peaks, one of which corresponds to an m/z value of 15. How can this reading be interpreted?

Answer 21

One of fragments has m/z or mass to charge ratio of 15 amu/charge
- remove one electron from methyl group > charge of +1 per every 15 amu

Question 22

Sandwich ELISA (Capture assay)

Answer 22

• analysts between 2 antibodies

Question 23

Mass spec utilizes what about magnetism

Answer 23

applying uniform accelerator electric field > uniform and constant veloicty

• path is related to mass and charge