BLG 888 - Western Blotting

What does Western Blotting detect

Proteins

What does Southern Blotting detect

DNA

What does Northern Blotting detect

RNA

What does Eastern Blotting detect

Post Translational modifications of proteins

What does it mean if the lac(i) is negative

The lac repressor is not functional

What does it mean if the lac(i) is positive

The lac repressor is functional

What does it mean if the lac(z) is positive

Can produce B-galactosidase, which converts lactose to allolactose, which is the inducer of the operon

What does it mean if the lac(z) is negative

Cannot produce B-galactosidase

What does it mean if the lac(y) is negative

Lack of Lactose permease therefore no transport

What does it mean if the lac(y) is positive

Lactose can be transported

What does LacA code for

Beta Galactosidase transacetylase, which adds acetyl groups to lactose

What does the LacI gene being expressed constitutively mean?

It will constantly be transcribed by cells whether lactose is present or not

Where does LacI bind and what is the effect of it

Binds to the promoter of the lac operon which prevents RNA polymerase from binding resulting in the rest of the lac operon to not be expressed.

what are the two conditions that allow for structural genes of lac operon to be expressed

1. LacI protein binds with allolactose, resulting in LacI is released from promoter Sequence

2. CAP is expressed and forms a complex with cAMP (meaning low glucose concentrations)

What happens if glucose levels are high

cAMP is low, which means CAP does not need to be expressed, therefore glucose level was high saving the energy from metabolizing difficult sugars such as lactose

Wht does CAP stand for and do

Catabolite Activator Protein is a transcription activator/enhancer

What is an Operon

Set of Genes that are regulated together and controlled by a single promoter

Promoter

Region of DNA that initiates transcription of a particular gene

Operator

Segemnt of DNA which a TF binds to regulate gene expression. This is where the repressor in Lac operon would bind

What does Allolactose do

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

Tris-HCl Buffer purpose

Buffering agent used for pH regulation

(SDS)/ Detergents

1. Ceel Lysis

2. Coating of proteins with negative charge

What is hte purpose of SDS Detergent

Take the protein from it’s native shape and open it up into a linear piece, which allows it go move through the gel

Glycerol

Makes the sample more dense than the sample buffer, so the sample will sit at the bottom

EDTA

Chealting agent which reduces oxidation damage to chelate metal ions

Mercaptoethanol

Reducing agent that disrupts disluphide bridges in proteins, resulting in protein monomers

PVDF

Polyvinylidene fluoride, non-reactive polymer, where it immobilizes proteins due to non specific affinity for amino acids

TEMED

Essential Catalyist for acruamide gel polymerization

What does altering the size of pores in the gel do

If you have a smaller size of protein, you use higher percentage of acrylamide/bis to let the smaller proteins seperate to the bottom and if you have a bigger size of protein then you use a lower percentage

How did we visualize the proteins

Coomassie Blue

Coomassie Blue Characteristics

1. Binds hydrophobically to the backbone of the protein molecules

2. Linear in binding to different proteins

3. Interact electrostatically but noncovalently with the amino and carboxyl groups of proteins

How is Electrophoretic Transfer performed

Placing the gel next to the membrane in a special casseste which is placed in a tank of electrophoretic buffer which conteains methanol.

What does methanol do in the electrophoretic buffer

1. Promotes dissociation of SDS from the protein

2. Improves adsorption of proteins onto membranes in the presence of SDS

Where does SDS bind

Binds to the positively charged and hydrophobic residues of proteins through its sulfate groups and alkyl chains

Monoclonal Antibodies

Generated by identical B cells and have monovalent affinity and recognize the same epitope of an tnaitgen

Polyclonal Antibodies

Mixture of heterogeneous which are usually produced by different B cells, and can recognize and bind to many different epitopes of a single antigen.

What was the secondary antibody in the experiment

Anti-mouse IgG Alkaline Phosphatase Conjugate

Explain how we get the dark blue precupitate

1. BCIP reacts with Alkaline Phosphatase

2. BCP and inorganic phopsphate which reacts with NBT

3. Resulting in Colour for visualization

TBST

Tris Buffer Saline and Tween 20 is a buffer for washing nitrocellulose membranes in Western Blotting, prevents non-specific binding of other proteins