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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 50 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

○ 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 Don't forget about the age old question of What is the meaning of sickle-cell disease?
We also discuss several other topics like What is the purpose of facilitated communication?

● #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 If you want to learn more check out What is the meaning of life outcomes data (l-data)?

■ Advantage: clear separation

○ Treat the cells with different salt conditions before doing equilibrium spin (in set up) to activate R 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?

■ 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 If you want to learn more check out What is the meaning of injustice?

difference in size without hsp90

● Antibody

○ Note: antibodies recognize antigens with specificity

■ 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 If you want to learn more check out What are the four components of sexual intelligence?

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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