PROTEIN BIOCHEMISTRY

Question 1

*How do you distinguish between the genome and the proteome?

Answer 1

Genome- set of genes present in a cell or organism
Proteome- set of proteins that are expressed and modified by cell. Proteome is NOT Constant unlike genome as it VARIES based on biochemical conditions.

Question 2

*What properties of proteins allow them to be purified from a crude cell extraction?

Answer 2

differences in chemical properties such as solubility, size, charge and specific binding affinity.

Question 3

*What is SDS and how does it work? How does it participate in gel electrophoresis?

Answer 3

SDS- Sodium Dodecyl Sulfate- detergent that denatures proteins (cause protein to unfold, become linear). Sodium aids in gel electrophoresis since it is negatively charged and will bind to + charge on protein, make all charges on protein negative.

Question 4

*Describe the principle of Isoelectric focusing?

Answer 4

process of separating proteins based on their amount of acidic and basic amino acids.
process- place a mixture of proteins in gel with proton gradient, apply electric field, and watch proteins migrate until at isoelectric point (pH at 0 net charge)

Question 5

*How can an antibody be used to specifically purify a protein?

Answer 5

Antibody- protein made in response to presence of foreign agent called antigen. Antibodies are able to recognize a particular structural feature of antigen called epitope.

Question 6

*Describe the distinction between a polyclonal antibody and a monoclonal antibody

Answer 6

monoclonal antibody- a cell makes antibody that recognizes ONE specific epitope on antigen.
Polyclonal antibody- DIFFERENT cells produce antibodies to an antigen with MULTIPLE epitopes.

Question 7

*Describe the steps in a western blot analysis.

Answer 7

Western blotting (immunoblotting)-

1. proteins separated in SDS-PAGE gel,
2. transfer proteins to polymer sheet (blotting)
3. primary antibody bind to protein of interest
4. wash, Stain with Fluorescent antibody; this secondary antibody(linked to enzyme for color) now bound to primary antibody
5. excite secondary antibody through light,
6. locate protein band detected by fluorescent emission of antibody.

Question 8

*What is required to occur before purifying proteins?

Answer 8

An assay or test that determines if the protein of interest is present.

Question 9

*Describe how protein purifications are monitored. What is relationship between specific activity and purification?

Answer 9

Monitored by determining specific activity of protein being purified. Specific activity- measure level of enzyme activity based on protein concentration. specific activity should increase along with each step of purification.

Question 10

*Explain the process of SDS-PAGE. What does it help distinguish about proteins?

Answer 10

Sodium Dodecyl sulfate Polyacramide Gel Electrophoresis- process of separating proteins bases on size. SDS will denature protein and make all charges on protein negative. Then a current will be run through gel and proteins will migrate to positive electrode on gel. Identify that small molecules move faster towards gel; larger molecules move slower.

Question 11

Describe how Affinity Chromatography works?

Answer 11

some proteins have a high affinity for a specific chemical or chemical group. Beads that have specific chemical group attached made. Protein mixtures will run through a column where only proteins that have an affinity for certain chemical group will be retained. The bound protein can then be released by passing a solution that is loaded with specific chemical that protein was attached to.

Question 12

How can proteins that are separated by SDS-PAGE be visualized (describe technique) ?

Answer 12

The Polyacramide gel can be stained with dyes like Coomassie blue that will present a series of band of different pigment levels (protein stain blue on clear surface).

Question 13

How can proteins be separated by gel electrophoresis? What info does SDS PAGE provide about proteins?

Answer 13

charged proteins are able to migrate in a gel with an applied electric field.
SDS PAGE- gives accurate determination of mass; SDS denatures proteins (1 SDS bind 2 aa) and protein will have same charge to mass ratio, only leaving them to migrate in gel based on mass only.

Question 14

In gel electrophoresis which molecules are the fastest to migrate down gel?

Answer 14

Smaller molecules will migrate faster. (Larger molecules are slow).

Question 15

What occurs in 2D Gel Electrophoresis?

Answer 15

proteins are separated in one direction by isoelectric focusing (protein migrate until reach pI), and then gel attached to SDS PAGE gel at 90 degree angle from isoelectric focusing separation (horizontally)

Question 16

What is the structure of antibody?

Answer 16

2 antigen binding sites, 2 heavy and 2 light chains in a Y- shape form.

Question 17

Describe the process of preparing monoclonal antibodies for a desired specify.

Answer 17

Since, antibodies do not last for a long time, they eventually die. Hence using immortal cell lines that produce monoclonal antibodies can be done with fusion of normal antibody producing cells with multiple myeloma cells (cancer). Then after fusion, one will screen for cell that make antibody of specificity and grow them in culture.

Question 18

Explain the example of how immortal monoclonal antibodies are created with rat experiment.

Answer 18

inject rat with antigen (contains specific epitope antibody binds to). remove spleen cells from rat uterus that contain antibodies for antigen. fuse those antibodies with myeloma cells in solution. grow hybrid cells and screen for cells that make specific antibody, grow in mass culture or inject cells in rat to induce tumor.

Question 19

What is ELISA and how does it work? How are antibodies helpful with this process?

Answer 19

Antibodies can be used a reagent to determine the amount of protein or antigen present.
ELISA- Enzyme Linked Immunosorbent Assay is a lab test to detect antibodies in sample.
process- detecting the presence and quantity of a protein since the antibody being linked to an enzyme to create a reaction that identifies colored product.

Question 20

Distinguish between Indirect ELISA and Sandwich Elisa. what do they have in common.

Answer 20

Indirect- coated antigen at bottom of well, specific antibody binds to antigen. another antibody that is linked to enzyme will bind to specific primary antibody, addition of substrate and enzyme converts it into colored product.
Sandwich Elisa- monoclonal ANTIBODY is coated and in bottom of well. antigen binds to antibody, second monoclonal antibody with enzyme binds to immobilized antigen and substrate added, converted into colored product by enzyme.
For both processes the extent of color formation, proportional to amount of antibody.

Question 21

What is the purpose of Mass spectrometry?

Answer 21

Mass spectrometry helps determine protein’s identity (mass to charge ratio).
If unknown protein in 2D gel can be removed and then protein can be cleaved and subject to MALDI-TOF, revealing peptides with known masses.
These masses can be compared to proteins in data base that are electronically cleaved by computer

Question 22

What are 2 forms of mass spectrometry that can be used to determine protein’s mass?

Answer 22

MALDI-TOF (Matrix Assisted Laser Desorption) Time of flight- analyze protein mass to charge ratio based on the time it takes for neg charged molecules to migrate time of flight tube or reach detector (proteins to matrix to be excited by laser)
ESI (electronic spray Ionization)- produce ions by applying high voltage to liquid and turning molecules into gas ions.

Question 23

In mass spectrometry, do lighter or heavier ions move faster (arrive at detector first)

Answer 23

Lighter ions move faster.

Question 24

What science technique can be used to determine primary sequence of protein?

Answer 24

Tandem Mass spectrometry- using 2 mass analyzers coupled together to increase ability to analyze chemical samples. Break down ionized precursor ion to product ions for detection.

Question 25

describes the kind of insight that primary structures can provide about protein.

Answer 25

primary structures from different proteins can infer knowledge about protein structure and function, evolution(by comparing similar proteins), history of individual protein (internal repeats), guide in exploring nucleic acid info, insight into molecular basis of disease and reveal presence of aa that regulate protein function/location.