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CAL / MCB / MCB 135A / What is the meaning of denatured?

What is the meaning of denatured?

What is the meaning of denatured?

Description

School: University of California Berkeley
Department: MCB
Course: Molecular Endocrinology
Professor: Gary firestone
Term: Fall 2018
Tags: Binding experiments, fractionation, immunoprecipitation, and westernblot
Cost: 25
Name: MCB 135A Week 2 notes
Description: Lecture notes from second week of class.
Uploaded: 09/03/2018
6 Pages 151 Views 4 Unlocks
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Lecture 3 (8/27/18) 


What is the meaning of denatured?



Reader pp. 17 - 21

Question on p. 21

Native conditions​: “natural”

● Receptors are functional and can bind to ligand/other proteins

Denatured

● Disrupted conformation; NOT functional

● Useful for measuring length of backbone

Fractionation Experiments 

● Gel filtration

○ Set-up: column containing beads with pores, put cytosol with *S through ■ Large molecules elute out first because they avoid the pores in the beads

■ Small molecules enter the pores and take longer to elute out of the column We also discuss several other topics like What caused malaria?

○ Graphed results would show two peaks (refer to #1 below)

■ First peak would show *SR and second peak would show *S


What is the meaning of gel filtration?



■ Quantify amount under peak to find amount of radioactivity

● #1: shows *SR and *S

● #2: shows a different receptor from #1 because the size is smaller ○ ALSO POSSIBLE: since these are native conditions, same size receptor but:

■ #1 R bound to extra ligand

■ #2 R is a half-sized receptor (and #1 is full-sized)

● #3: Rec- cell line

● Sucrose gradient


What is the meaning of sucrose gradient?



○ Set-up: tube with low/high gradient, do equilibrium spin to separate molecules by density

■ Take cytosol, add *S, put through sucrose gradient, *S and *SR will separate bc different size

■ Advantage: clear separation

○ Treat the cells with different salt conditions before doing equilibrium spin (in set up) to activate R We also discuss several other topics like What is the purpose of facilitated communication?

■ Note: physiological salt concentration = 150 mM

■ Low salt conditions: < 150 mM

● In low salt conditions R is bound to hsp90 (inactive)

● Sucrose gradient result: *SR separates further from *S because there is a bigger difference in size due to hsp90

■ High salt conditions: > 150 mM

● High salt condition activates R

○ Disrupts hydrogen bonding between R and hsp90

● Sucrose gradient result: *SR is closer to *S because smaller

difference in size without hsp90

● Antibody

○ Note: antibodies recognize antigens with specificity We also discuss several other topics like What is the meaning of life outcomes data (l-data)?
If you want to learn more check out Who believed that man had to participate in the carry out of the government and that man is political by nature?
If you want to learn more check out What is hylomorphism?
We also discuss several other topics like What are the four components of sexual intelligence?

■ Example: Anti-X Ab would recognize X but not Y and vice versa ■ Assumption: antibody has no effect on steroid bonding to R

○ Set up: add *S to cytoplasm (R+), covalently bind Ab to bead, Ab binds to R which is bound to *S, centrifuge

■ Pellet: bead + antibody + receptor + *SR

■ Supernatant: other receptors not recognized by Ab, *S

Lecture 4 (8/29/18) 

Reader pp. 18 - 23

Question p. 21/p. 23

Antibody Specificity

● High affinity binding

● Negative control: run beads only (without anti-receptor antibody) ○ Ensure that measured radioactivity isn’t just radioactivity trapped in beads

Q: Test whether *Sx binds estrogen receptors 

A: Use cells that express estrogen. Homogenize cells (spin 100,000 x g spin), use supernatant

● Positive control (A): cytosol (ER+ aka under native conditions), add radioactively marked estrogen, add Bead-Anti-ER

○ Negative control (B): same set-up as (A) but bead only

○ If results show significant radioactivity relative to negative control (B) = shows that you can detect binding to ER under the conditions of the experiment

● (C): experimental result with cytosol (ER+), *Sx, and Bead-Anti-ER

● Negative control (D): use cytosol (ER+), add *Sx, add beads (without Anti-ER) ● Using positive + negative controls, you can unequivocally conclude that the unknown steroid binds to ER

Glass Fiber Filter

● Put cytosol on filter, wash it, *S drain away and *SR are bound to glass ● Discovered by accident when researchers realized receptors bind to glass

Gel Filtration

● Native conditions: different sizes can mean either:

○ (1) Rx (ligand attached) vs R OR

○ (2) R vs ½ R (at least partly functional)

○ Can’t conclude anything further because experiment is under native conditions

● Run a Western blot​ under denatured conditions 

○ Cells → Cell extract → (use SDS-Polyacrylamide Gel) → Electrophoresis ■ Typical cell has 10,000 - 20,000 proteins, so you have a mess when you denature everything

○ Make a replica of gel and transfer to membrane, add primary Ab-ER, add secondary Ab bound to enzyme that converts substrate into product ■ Secondary Ab binds to primary Ab and amplifies signal

■ Conversion of substrate → product can be detected by x-ray film

○ Results show endogenous size of the receptor

■ Denaturation dissociates R from any ligands it is bound to

■ IF receptors are same size: gel filtration showed same receptor

■ IF receptors are different size: gel filtration showed two different receptors of different sizes

Lecture 5 (8/31/18) 

● Kd: dissociation constant aka measure of affinity

○ Note: measure of affinity always refers to dissociation/“pulling off”, NOT association!

Binding Assays: separate *S from *SR 

● Native conditions

○ Whole cell assay

○ Size separations: gel filtration, sucrose gradients

○ Antibodies-- specificity using Anti-Rec

○ Glass fiber assay: property differences

● Denatured conditions: determine size of rec proteins

○ Western (protein) blots

■ Electrophoretic Gel (SDS-PAGE)

■ Blot onto polymer sheet

■ Primary antibodies + secondary antibodies

■ Result: see intrinsic side of polypeptide backbone

Q: How do you distinguish between 3 cells-- 1 WT SR, 1 point mutation (AA substitution; does not bind steroid), 1 Rec- cell line 

A: Use binding assay using radiolabeled steroid under native conditions to distinguish WT from non-WT cell lines-- BUT can’t distinguish between mutated and Rec- cell lines ● Use Western blot and treat with Anti-Rec

○ Mutated cell line would have same size as WT because amino acid substitution doesn’t change length of polypeptide chain

○ Rec- cell line wouldn’t show up

○ Note: under denatured conditions, antibodies attach very well to proteins (so we can still use them to identify specific receptors)

Immunoprecipitation 

● Treat bead with Anti-Rec, treat with cell extracts without steroid, spin down to get pellet with beads (attached to protein of interest) and supernatant (contains all other proteins in cell)

○ Denatured condition: put in SDS-PAGE, probe with 1˚ antibody, examine under x-ray film

■ Denaturing dissociates antibody from receptor

● Negative control: beads only (without antibody)

○ Ensure that radioactivity isn’t simply getting trapped in the beads Co-immunoprecipitation

● Detect protein-protein interactions, i.e. check whether X is bound to Z ● Beads bound to anti-X, add cell extracts (native condition), spin to get pellet, denature pellet, put in SDS-PAGE and do Western blot

○ In Western blot, treat with primary antibody (Anti-Z) to check whether Z is in the pellet

■ IF X is bound to Z, then Bead-Ab-X-Z would be in pellet

■ Can also treat with Anti-X to make sure beads were correctly bound to X

● Confirms that experimental set up was correctly done

■ If you think Z is there but not showing up, you can run a Western blot with the cell extracts to confirm that Z is expressed but does not bind X

● Negative control: beads only (without antibody)

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