day 2: protein techniques + enzymes

Question 1

proteome

Answer 1

like a genome, but for proteins (Proteome: function of all proteins)

Question 2

proteins can be purified based on:

Answer 2

net charge

size

sedementation rate

solubility

binding capabilities

Question 3

how do you liberate materials from a cell? (to find proteins of interest)

Answer 3

sonication or homogenation

Question 4

once you’ve liberated material from cells what do you want to do?

Answer 4

separate out the proteins from each other to find protein of interest

Question 5

how do you separate proteins?

Answer 5

9 techniques

Question 6

how does separating proteins work

Answer 6

Separation methods target the differences in protein structures such as charge, size, solubility, and specificity of binding sites or active sites. The “fractions” are collected and assayed

Question 7

9 techniques for separating proteins

Answer 7

1. Salting out
2. Gel filtration chromatography
3. Ion exchange chromatography
4. Affinity chromatography
5. Polyacrylamide Gel Electrophoresis (PAGE)
6. SDS
7. Isoelectric focusing
8. Density gradient centrifugation
9. immunoprecipitation

Question 8

the 3 steps for getting to your identified protein

Answer 8

1. Release all proteins from the cells
2. Separate types of proteins from each other (fractionation)
3. Identify which fraction contains the protein of interest (assay)

Question 9

identification assays

Answer 9

1. Enzyme activity if the protein is an enzyme (e.g., colorimetric assay)
2. Bioassays if the protein has a known effect on a tissue (e.g. slows heart rate)
3. Antibody (Ab) binding with an antibody generated in response to that protein (note: Ab is covalently attached to a fluorescent or radioactive molecule for detection)
4. Ligand binding if the specificity of the protein’s binding site is known (e.g. the lectin, Con A binds glucose or estrogen receptor binds estrogen. The ligand is made radioactive for detection)

Question 10

how would you go about characterizing the purified proteins?

Answer 10

The protein can be digested into amino acids to determine the composition (the percent of each different amino acid in the protein)

2. The sequence of amino acids can be determine using stepwise removal of each amino acid by Edman degradation or by using tandem mass spectroscopy
3. Xray crystallography shows 3-D shape
4. 2-D NMR techniques allow some features of 3-D

Question 11

How to break open cells:

Answer 11

1) You’d literally get a beef liver, put it in a blender. Keep it cold, blend extremely short time- got ‘crude extract’ aka starting material
2) Might also use the homogenizer tube; glass tube, mortar + pestle style, smushes cell membranes down
3) Last way: sonicator. Uses sound waves.

Bcaterial cells super hard to open up – use a french press. Plant cells too

Question 12

Differential centrifugation

Answer 12

Cells are disrupted in a homogenizer and the resulting mixture, called the homogenate, is centrifuged in a step-by-step fashion of increasing centrifugal force. The denser material will form a pellet at lower centrifugal force than will the less-dense material.

Most dense is nuclear stuff. Next most dense is mitochrondria. Then microsomal. Then cytosol.

Question 13

Cytosol:

Answer 13

all soluble components of cell that are not particulate. Aka all dissolved proteins, free ribosomes, all soluble portion of cytoplasm.

Question 14

how do u remove salt after salting out

Answer 14

dialysis

Question 15

Gel filtration chromatography

Answer 15

•separates protein according to size- called gel filtration. The big protein cant fit in the little pore– goes straight down. Intermediate protein can fit in some but not all pores, some sinks down, some stays. Little guy fits right into the pores of the beads, stays on top. Little proteins exit the column last because they can hang out in the pores of the beads.

Question 16

Ion exchange chromatography

Answer 16

separation of proteins on the basis of charge. The beads in the column have a + or - charge

Question 17

Affinity chromatography

Answer 17

takes advantage of the fact that some proteins have a high affinity for specific chemicals or chemical groups. Beads are made with the specific chemical attached. A protein mixture is passed through the column. Only protein with affinity for the attached group will be retained.

Question 18

native gels vs denaturing gels

Answer 18

Proteins will migrate in an electrical field because they are charged. When the migration occurs in a gel, the process is called gel electrophoresis. This can either a “denaturing gel” with SDS as the denaturant or a “native gel”.

Native gels separate by charge and mass and preserve the functional protein shapes.

denaturing migrate based on SIZE ONLY

Question 19

SDS separates proteins based on:

Answer 19

size

Question 20

Isoelectric focusing

Answer 20

allows separation of proteins in a gel on the basis of their relative amounts of acidic and basic amino acids. If a mixture of proteins is placed in a gel with a pH gradient and an electrical field is applied, proteins will migrate to their isoelectric point, the pH at which they have no net charge.

Question 21

Density Gradient Centrifugation

Answer 21

Ultracentrifugation can be used to examine proteins. When subjected to a centrifugal force, the rate of movement of the particle is defined by the sedimentation coefficient, s. When centrifugation is through increasing densities it is called Density Gradient Centrifugation

its a way of separating out proteins from each other- one of the methods of fractionation

Question 22

Identification assays enable scientists to:

Answer 22

locate which fraction has the protein of interest using assays for activity or binding ability

Question 23

Immunological techniques can be used to both purify and identify proteins

Answer 23

The estrogen receptor binds the steroid hormone estradiol tightly and with great specificity.

The estrogen receptor has no enzymatic activity, but can be purified by immunological techniques and the use of gradient centrifugation

Question 24

can you generate antibodies 2 specific proteins?

Answer 24

yes

Question 25

An antibody is a protein synthesized in response to the presence of a foreign substance called an ____

The antibody recognizes a particular structural feature on the ___ called the ___

Answer 25

antigen

epitope

Question 26

Antibodies are proteins secreted by ___after stimulation from a foreign shape

Answer 26

mature B lymphocytes

Question 27

Virgin b cell

Answer 27

has not yet been triggered by an antigen. Still has antibody cell attached to its membrane. Now the antigen comes by- if it bumps into that antibody and binds, bc it matches the shape, then it triggers an immune response in that particular virgin B cell. That virgin b cell now divides into many cells and starts secreting that antibody instead of just having it attached to its cell surface

Question 28

T/F: DNA varies between different virgin B cells

Answer 28

true

Question 29

Polyclonal antibodies

Answer 29

A Polyclonal Antibody represents a collection of antibodies from different virgin B cells that recognize multiple epitopes on the same antigen. Each of these individual antibodiesrecognizes a unique epitope that is located on that antigen.

• are isolated from the blood of an animal challenged with an antigen
• vary in amino acid sequence from each other
• may recognize more than one epitope on an antigen

Question 30

Monoclonal antibodies

Answer 30

While Polyclonal antibodies are heterogeneous mixtures of antibodies, monoclonal antibodies are all identical, produced by clones of a single antibody-producing cell. They recognize one specific epitope

Question 31

Purification by immunoprecipitation

Answer 31

Monoclonal antibody to the estrogen receptor is added to a preparation of cytosol containing the receptor. The antibody is attached to an insoluble bead. The mixture is then gently stirred for several hours to allow the interaction of the receptor and antibody. The mixture is centrifuged and the supernatant is discarded. The pellet is resuspended in buffer to wash out any trapped cytosolic components. The pellet is again collected and the supernatant discarded. The antibody–receptor complex is treated with a denaturant. On centrifugation, the supernatant contains pure estradiol receptor.

• centrifuge
• antibody bound 2 beads
• collect pellet that has antibodies in it

Question 32

can immunological techniques only purify, or also identify proteins?

Answer 32

both

Question 33

immmunological protein identification technique

Answer 33

ELISA

Antibodies are used as a reagent to quantify the amount of a protein or other antigen. Enzyme-linked immunosorbent Assay (ELISA) quantifies the amount of protein present because the antibody is linked to an enzyme whose reaction yields a readily identified colored product.

Question 34

western blotting is an example of using antibodies for what purpose?

Answer 34

protein identification

In western blotting or immunoblotting, proteins are separated in an SDS gel, transferred to a polymer, and then stained with a fluorescent antibody specific to the protein of interest

Question 35

what’s the point of isolating proteins?

Answer 35

once isolated, proteins can be characterized- you’re trying to find its amino acid sequence (primary structure)

Question 36

what are some methods to determine amino acid sequence?

Answer 36

edman degradation

mass spec

Question 37

why do we care about the amino acid sequence?

Answer 37

1. Different proteins can be compared to infer knowledge about structure and function.
2. Comparison of similar proteins from different species provides information about evolution.
3. Primary structure can reveal the presence of amino acid sequences that regulate protein function and location (e.g. signal sequences)
4. Comparison with the normal protein sequence can provide insight into the molecular basis of disease.
5. Primary structure can be used as a guide to find its gene by reversing the translation code to create probes

Question 38

why do we care about 3d structure

Answer 38

(X-ray Crytallography) to determine the 3-D structure. 3-D structure reveals motifs that reveal function such as the four calcium binding sites in Calmodulin.

Question 39

Raising Monoclonal antibodies

Answer 39

1. Inject mouse with foreign protein of interest and wait 4- 8 weeks.
2. Remove the spleen and isolate the B lymphocytes
3. Fuse the B cells with an immortal myeloma tumor cell type to make each B cell immortal in cell culture. Test which clones recognize the antigen and culture each selected B cell clone in a different culture plate.
4. Isolate the supernatant of the cell culture to isolate a single type of antibody recognizing one epitope on the antigen.
5. Monoclonals give reliable data because there are no host antibodies to confound the results. However, the detection is more difficult because the signal from a fluorescent tag on the Ab is several times weaker than polyclonals which have several different epitopes bound to one antigen.