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Cell Bio Lab quiz 2


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How treatments to cells change protein expression
LPS: increases protein expression
LTA: increases protein expression
Na3VO4: decreases protein expression

Regulation by Kinases and Phosphotases
-Kinases add phosphate molecules to proteins on serine, threonine, or tyrosine AA residues (activate)

-Phosphotases remove phosphate molecules from proteins on serine, threonine, or tyrosine AA residues

Lipopolysaccharide (sugar backbone)
-comes from gene(-)bacteria on the walls
-acts as an endotoxin, causing a STRONG IMMUNE RESPONSE
-Binds to Toll-like Receptor4 (TLR4)-recruits adaptor molecules
-Adaptor molecules ACTIVATE kinases which amplify the signal-INFLAMMATORY RESPONSE

Lipoteichoic Acid
-From gene(+) bacteria walls
-acts as an endotoxin, causing a STRONG IMMUNE RESPONSE
-Binds to Toll-like receptor2(TLR2)
-TLR2 recruits adaptor proteins to ACTIVATE kinases
-Kinases amplify the signal causing INFLAMMATORY RESPONSE

Sodium Orthovanadate
-An inhibitor of protein phosphatases

Western Blottin Defined
Analytical technology used to detect specific proteins in a given sample of tissue homogenate or extract
Western Blotting Steps
Blot Blocking
Primary AB

Sorts protein based on SIZE
-Anionic detergent(-) that disrupts 2 and 3 structure to apply a uniform (-) charge
-Higher (-) = bigger protein

Western Blot Transfer
Transfer from gel to PVDF membrane
-Transfer it outwards
-Needs a transfer buffer

Blot Blocking
Milk formula blocks non-specific protein binding sites
-If we dont block -- primary antibody will bind to everything
Protein of Interest
-PAB will bind to STAT3
Secondary Antibody
2 anditbody is specific to 1 antibody
-2AB is flourescently labeled (2*)

ECL Chemiluminescence
(Like calcien AM)
-Light sensitive reagent - allows us to visualize the flourescence emitted by secondary AB

Image is generated to which differing intensity of flourescence can be visualized
-More dense of a band, the more protein of interest
-Higher intensity=higher protein of interest

Overall Steps
1.Treated the cells (RAW264.7 mouse cancer cells)
2.Lyced the cells - lets the proteins out
3.SDS-PAGE - sort proteins by size (PVDF membrane)
4.Block proteins not of interest (blockers applied)
5.Primary Antibodies attach to STAT3(protein of interest)
6.Secondary Antibodies attach specifically to primary antibodies

TLR4 Complex

-Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
-Lipoteichoic Acid

What is Densitometry
a technique used to give us quanititative data from a picture
-Numerical value based on amount of pixels that are darkened versus the amount that are not
-the darker the band the higher the number

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