Experiment 4, 5 and 6

Question 1

What technique do you use to find total protein content?

Answer 1

Measure A280 using a spectrophotometer

Question 2

What technique do you use to find the molecular weight of a protein?

Answer 2

SDS PAGE

Question 3

What technique do you use to quantify a specific protein?

Answer 3

Western Blot and staining with a specific antibody

Question 4

Within a cell, what goes on regarding EGF?

Answer 4

EGF binds to EGF receptor (EGFR)
Signal from EGF receptor is transmitted to the nucleus
Turns on genes

Question 5

What is the signaling from the EGF receptor?

Answer 5

Kinases
–> phosphorylated other proteins
–> can turn on, or turn off these proteins
–> phosphorlate at tyrosine amino acids or serine/threonine amino acids

Question 6

The EGF receptor is actually a ______

Answer 6

receptor tyrosine kinase

Question 7

receptor tyrosine kinase

Answer 7

–> phosphorylates itself and other intracellular signaling proteins at tyrosine amino acids
–> phosphotyrosines create binding sites for other proteins
–> this tyrosine phosphorylation (pTyr) starts the signaling pathway (allows other proteins to bind to the EGF receptor)

Question 8

What activates transcription factors?

Answer 8

Signaling pathways

Question 9

Steps for SDS page and Western Blotting

Answer 9

1. cell disruption
2. determination of protein concentration
3. denature proteins
4. electrophoresis of proteins
5. determine the molecular weight of the unknowns

Question 10

Why do we use detergents?

Answer 10

To solubilize membranes, solubilize integral membrane proteins and stop cellular function

Question 11

What detergent did we use in the experiment?

Answer 11

Sodium Dodecyl Sulfate (SDS)

Question 12

What does SDS do?

Answer 12

–>strong ionic detergent
–> quickly solubilizes the cell
–> denatures proteins
–> neutralize the charges of the amino acid side chains
–> overall negative charge
–> creates a univform charge-to-mass ratio

Question 13

What happens when cells lyse?

Answer 13

Solubilizing cells releases proteases sequestered in specific parts of the cell. Proteases can destroy your sample proteins. So, proteases must be inhibited if using extensive manipulation of samples. In our experiment, we use ice to slow down that process

Question 14

In preparing your samples for SDS-PAGE, you added sample buffer that contained gylcerol and bromophenol blue. What is the purpose of the glycerol?

Answer 14

increase the samples density. This is so it sinks to the bottom of the loading well and to keep it from diffusing into the running buffer

Question 15

In preparing your samples for SDS-PAGE, you added sample buffer that contained gylcerol and bromophenol blue. What is the purpose of the bromophenol blue?

Answer 15

used as a dye so that you can see the proteins go through the gel.

Question 16

How do you estimate protein levels?

Answer 16

Warburg-Christain method

Question 17

Warburg and Christain Equation

Answer 17

Estimates protein levels
Equation: Protein (mg/ml) = (A280) x (Correction Factor) x (DF)

Question 18

A280

Answer 18

Absorbance of your sample

Question 19

How do you determine the number for correction factor?

Answer 19

A280/A260

Question 20

What is the correction factor?

Answer 20

1.12

Use if A280/A260 ratio is greater than 1.7
If A280/A260 ratio is less than 1.7, must use table

Question 21

DF

Answer 21

Dilution factor of the dilution you made of your sample

Question 22

Why do we standardize the protein amount?

Answer 22

Each sample must have the same amount of protein

Question 23

How do you denature the proteins?

Answer 23

–> SDS
–> 2-mercaptoethanol (breaks disulfide bonds)
–> boil the samples)

Question 24

For SDS-PAGe we must separate proteins in a _______ _______.

Answer 24

polyacrylamide matrix

Question 25

How do we prepare the polyacrylamide matrix?

Answer 25

1. Chemically cross-linked acrylamide and bis-acrylamide 2. TEMED and Ammonium Persulfate (AP) initiate the cross-linking

Question 26

To chemical cross-link acrylamide and bis-acrylamide to form the polyacrylamide gel we used 2 reagents:

Answer 26

AP and TEMED

Question 27

Due to the ring structure of the amino acids ________________________, _____________________, __________________, proteins can absorb ultraviolet light

Answer 27

phenylalanine, tyrosine and tryptophan

Question 28

How do the proteins move in electrophoresis?

Answer 28

Proteins (negative chrage) migrate to the postive pole

Question 29

Discontinuous buffer system

Answer 29

Stacking gel-4% acrylamide Resolving gel- 12% acrylamide Different ions in the gel (chloride ions) and the running barrier (glycine ions) Small proteins move easily through the matrix, but large proteins are held back Proteins run into the running gel Discontinuosu buffer front (chloride vs glycine) Result: proteins run through the resolving gel as a compressed band

Question 30

How do you transfer proteins from SDS PAGE to Western blotting

Answer 30

Lay a sheet of nitrocellulose onto the gel Electrophorese the proteins onto the nitrocellulose membrane the proteins stick and form an exact copy of the PAGE gel

Question 31

How do you make antibodies?

Answer 31

Purify protein Inject into animal Bleed animal after month or so Purify antibody from the blood

Question 32

Won't antibodies stick non-specifically to the nitrocellulose?

Answer 32

Block the blot so antibodies will not-specifically stick o the membrane Incubate blot with Bovine Serum Albumin Now no othe protein can stick to the blot

Question 33

True or false: secondary antibodies must be from a different species that primary antibody

Answer 33

True

Question 34

If primary Ab is Goat anti-...... then what must the secondary antibody be?

Answer 34

Rabbit anti-goat Ig Mouse anti-goat Ig Etc.

Question 35

What enzyme to use to label the secondary antibody? They are easily isolated in large quantities.

Answer 35

Horserash Peroxidase (HRP) Alkaline Phosphatase (AP) (what we used in class)

Question 36

What was detected when doing the Western Blot?

Answer 36

Tyrosine phosphorylated proteins over different amounts of time

Question 37

What would the appearance of a new band mean in a Western Blot?

Answer 37

Protein became phosphorylated at tyrosine residues. Does not mean a new protein has appeared.

Question 38

What does it mean when a band on the blot increases in density over time in Western Blot?

Answer 38

Proteins became phosphorylated at tyrosine residues

Question 39

What direct process in the cell would possibly account for the density of a band increasing in our experiment in Western Blot?

Answer 39

The activation of tyrosine kinases are phosphorylated

Question 40

What does it mean when a band on the blot decrases in density over time in Western Blot?

Answer 40

Protein becomes dephosphorylated or protein destruction occurs

Question 41

What direct process in the cell would possibly account for the density of a band decreasing in our experiment in Western Blot?

Answer 41

Activation of phosphatase enzymes

Question 42

Immunoglobulin

Answer 42

generic name for the antibody as a protein

Question 43

Antibody

Answer 43

refers to an IG that binds to specific antigen

Question 44

IgG

Answer 44

Main class of antibodies found in blood 20% of all plasma proteins, produces during secondary response Protects the body

Question 45

IgA

Answer 45

Found in large amounts in body secretions (tears, etc.) 2 forms: blood form and non secretory Secretory form: found in secretions has 2 monomer units, held together by a joining chain with an attached secretory component-- protects from protelytic enzymes

Question 46

IgE

Answer 46

Found mainly in body fluids and skin Has special affinity for receptors on plasma membrane of basophils (blood) and mast cells (tissue) Play a damaging role in allergy development

Question 47

IgD

Answer 47

Found only on the surface of B cells as B cell receptor (BCR) and is co-expressed with IgM

Question 48

IgM

Answer 48

Found as a monomer on the surface of B cells and is secreted as pentamer in plasma cells