BLG888: Test Test Lab 1 Review Western Blotting

When was DNA discoevered and by whom?

DNA was discovered on 1953 by James Watson and Francis Crick

What does a Western blot detect?

protein

What does a southern blot detect?

DNA

What does a norther blot detect?

RNA

What was the objective of the first experiment?

- Extracted cellular proteins from 2 different E.coli strains

- Identified which produces b-gal using SDS-PAGE and anti-b-galactosidase monoclonal antibodies

Which strain produces b-gal?

- EMG9

- EMG26 did not produce b-gal

What were the main steps of the first experiment?

- Isolate cellular proteins from two E.coli strains (one produces b-gal)

- Proteins extracted will be subjected to SDS-PAGE to produce a protein profile based on molecular weight

- One gel will be steined with Coomassie blue to show the presence of proteins

- The proteins of the other gel will be transferred to nitrocellulose filter by electrophoretic transfer

- b-gal is visualized on the nitrocellulose membrane using anti-b-gal monoclonal antibodies which conjugate to anti-IgG alkaline phosphatase conjugate

What are the different genes in the lac operon?

- lacZ

- lacY

- lacA

- lacI

What's the function of the lac operon?

to digest the monosaccharide lactose

What does lacZ do?

- it encodes the protein b-gal

- n-gal breaks lactose into galactose and glucose

What does the lacY gene do?

- Encodes lactose permease

- This membrane bound transport protein pump lactose into the cell

What does the lacI gene do?

- Encodes for the repressor protein that binds the operator region to inhibit transcription

What does the lacA gene do?

- Encodes for b-gal transacetylase, an enzyme that adds an acetyl groups to lactose and other galactose containing sugars

- Not well understood

What's CAP?

Catabolic activator protein

- it's a transcription activator that increases the rate of transcription

What's an operon?

Structural genes,, together with promoter and operator or activator binding sites

- Multiple genes that get transcribes together found in bacteria

What's the promoter region?

- A region of DNA that initiates transcription of a particular gene

- An RNA pol must bind that region to begin mRNA transcription

What's the operator region?

- Contains the binding site for repressor protein I

- When the repressor is bound to the operator RNA pol cannot bind to the promoter region

What's a repressor protein?

A DNA or RNA binding protein that inhibits the expression of one or more genes by binding to the operator

What is the function of b-gal?

- It has two functions

- Breaks lactose into glucose and galactose

- Converts lactose to allolactose which is an inducer of the operon

- Allolactose interacts with the lac repressor and causes the repressor to change to an inactive shape that is unable to bind to the operator site

the inactive repressor leaves the DNA and transcription starts

What does SDS (sodium dodecyl sulfate) buffer do?

- Breaks the 3d and 2d structure of proteins and linearizes them

- Coats the proteins with a negative charge

- When SDS is added proteins are distinguished by their molecular weight only and not their charge

What are detergents?

Molecules that manipulate (disrupt or form) hydrophobic-hydrophobic interaction

- Detergents are used to lyse cells and solubilize membrane proteins

- Denaturing deteregnets can be anionic such as SDS

What's running dye?

- The dye that allows the investigator to track the progress of the electrophoresis

- Eg. Bromophenol blue is often used, it has a negative charge so it migrates with the DNA

What are the parts of the lysis buffer used?

- Glycerol: makes sample more dense so the sample will remain in the bottom of the well rather than float out

- EDTA: is a chelating agent (reduces oxidation damage and to chelate metal ions, this inhibits some proteases)

- Marcaptoethanol: reduces (disrupts) disulfide bridges

What's tetramethylethylenediamine (TEMED)?

Catalyst for acrylamide/bis-acrylamide gel polymerization

What's TEMED used with?

- Ammonium persulfate (APS)

- Which catalyzes acrylamide polymerization when preparing for gel electrophoresis

What's a polyacrylamide gel?

-Polyacrylamide gels are three dimensional networds of acrylamide reacted with the bifucntional reagent Bis via a free radical initiated polymerization mechanism

How is the pore size of the gel determined?

- Pore size of the gel is directly related to the ratio of acrylamide to Bis

What's %T?

- Concentration of the gel monomers

- Pore size decreases as %T increases

What affects the pore size other than the %T?

- Cross linkage also affects the pore size

- As cross-linking agent (bis) concentration relative to the total monomer increases up to 5% by weight decreases the pore size

- Above 5% C, the pore size increases again because the cross-linking agent dimerizes with itself to form more expanded gels

How do you determine the pore size of the gel depending on the protein of interest?

- The smaller the size of the protein of interest the higher the percent of acrylamide/bis

- The bigger the size of the protein of interest the lower the percentage of acrylamide/bis

What are the types of gels in PAGE?

- Stacking gel

- Running gel

What's the stacking gel?

- The top part of the gel is the stacking gel

- It has a low percentage of acrylamide

- We don't want protein separation here

- Usually has a lower pH (6.8)

What's the primary structure?

Linear amino acid sequence of a protein polypeptide chain

What's the the secondary structure?

Local spatial conformation of the polypeptide backbone

- a-helices, b-sheets, and turns

What's the tertiary structure?

Three dimensional arrangement of all the atoms of the protein molecule

What's a discontinuous buffer system?

- Discontinous simply means that the buffer in the gel and the tank are different

- The system is set up with electrode buffer -> Stacking gel -> running gel -> electrode buffer

What's the function of the stacking gel?

It ensures that all the proteins arrive at the running gel at the same time , so proteins of the same molecular weight will migrate as tight bands

How does Coomassie blue bind to proteins?

- Binds hydrophobically to the backbone of the protein molecule

What is the electroblotting technique?

- Electrophoretic transfer is performed by placing the gel next to the membrane in a special cassette that in turn, is placed in a tank of electrophoretic buffer

- The transfer buffer contains methanol

- Upon application of voltage, the sample migrates out of the gel and onto filter paper

What's the purpose of methanol in the transfer buffer?

- Promotes dissociation of SDS from the proteins

- dramatically improves adsorption of proteins onto membranes in the presence of SDS

What is the set up of the elctrotransfer?

Cathode -> filter paper -> gel -> nitrocellulose -> filter paper -> anode

Is the anode positive or negative?

positive

What are monoclonal antibodies?

- Antibodies produced from a single group of genetically identical B-cells

- Have monovalent affinity and only recognize the same epitope (antibody binding site) of an antigen

What are polyclonal antibodies?

- Mixture of heterogenous which are usually produced by different B cell clones in the body

- Can recognize an bind to many different epitopes of a single anitgen

What was the primary antibody used in the experiment?

anti-b-galactosidase mouse monoclonal antibody

What was the secondary antibody used in the experiment?

anti-mouse IgG alkaline-phosphatase (AP) conjugate

What will the second antibody be attached to?

Alkaline phosphatase (AP)

What will the protein-antibody-antibody-alkaline phosphatase complex react with?

5-bromo-4-chloro-3-indolyl phosphate (BCIP) which is a substrate of AP in conjugate with NBT (nitroblue tetrazolium)

What's the function of Tween 20?

- Non-ionic surfactant used as a detergent

- Prevents non-specific binding of other proteins to the surface of the membrane