experiment 1 - beta-galactosidase protein using the western blot

western blot

detection of proteins

southern blot

detection of DNA

northern blot

detection of RNA

eastern blot

involves different types of probes to detect post-transcriptional modifications of proteins

lac operon enzyme encoding structural genes

• lac Z: encodes B-galactosidase enzyme (cleaves galactose and glucose so they can become allolactose)

• lac Y: encodes lactose permase

• lac A: encodes beta-galactosidase transacylase (adds acetyl groups to lactose and other galactose-containing sugars)

role of allolactose

binds to lac repressor and causes a conformational change to inactive shape so that it cannot bind to the operator site and transcription can occur

lac operon regulatory elements

• repressor I; lacI which encodes repressor

• promoter

• operator

purpose of Tris-HCl buffer in lab

• at pH 6.8

• used for pH regulation

purpose of lysis buffer

• stabilize proteins containing SDS

SDS

• what is it?

• role?

• sodium dodecyl sulfate

• anionic detergent

• role is cell lysis and coating of proteins with negative charge

what is PDVF

“Polyvinylidene fluoride or polyvinylidene difluoride” and it is is a highly non-
reactive polymer that is used as the membrane material in western blots, where it immobilizes proteins, due to its non-specific affinity for amino acids

using gel electrophoresis, how are molecules separated?

• charged molecules are separated via size and charge

• more highly charged species move more rapidly in an electric field

• larger molecules and those less spherical in shape are retarded by the gel to a greater extent

how does pH effect gel electrophoresis

• electrophoretic mobility of a protein is a strong function of pH in the range of pH 3-10, especially in the range close to its isoelectric point

in the pH between their isoelectropoints, how will 2 proteins move?

the two proteins will move in opposite directions

what are Polyacrylamide gels

three-dimensional networks of acrylamide

what is the strategy for altering pore size for your protein?

• The smaller the size of the protein of interest, the higher the percentage of
  acrylamide/bis.

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

what is the relationship between pore size and concentration

• the pore size of polyacrylamide gels (which are polymerized as the gel is formed) decreases as the concentration of the gel monomer (expressed as %T) increases

how is pore size related to cross linking?

• increasing the concentration of cross-linking agent relative to the total monomer (expressed as %C) 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

what does the movement of charged species through an electric field depend on?

• species net charge

• molecular radius

• magnitude of the applied field

how would proteins migrate in their native states?

• net charge or molecular radius of natively folded proteins is not weight dependent

• so in their native states, different proteins with the same molecular weight would migrate at different speeds in an electrical field depending on their charge and 3D shape

on what types of gels are most proteins fractionated on

polyacrylamide gels ranging from 5%T to 20%T, containing 2-5%C

when are non-denaturing buffers required?

required whenever biological activity must be retained

• also used when charge differences are known to exist which may give greater resolution than separation on the basis of size

when using a denaturing buffers like SDS what is the relevant information?

• only molecular size is relevant because proteins get separated

what does SDS do?

• it is an anionic detergent

• it unfolds the native protein → linearizes it

• binds strongly to positively charged and hydrophobic residues of proteins using its sulfate groups and alkyl chains

• confers a charge proportional to the length of the polypeptide chain

• pH is not important

explain the variation in charge density and electrophoretic mobility of proteins using SDS

• the charge density and electrophoretic mobility are nearly constant for most proteins

• exceptions are glycoproteins and highly basic nucleoproteins

what is a continuous buffer system?

• what is is used for

• what is its main disadvantage?

• same buffer is used throughout the gel at both electrodes

• require only a single layer of gel

• simple to set up

• used for monitoring an enzyme purification or for preparative electrophoresis of a partially purified protein

• main disadvantage is that the sample must be in highly concentrated form since diffusion is not counteracted in the system

what is a discontinuous buffer system?

• what is is used for

• concentrates each sample component into a narrow band known as the “stack”

• the original sample may therefore be much more diluted and components which band close together can be resolved more easily

• uses buffers of different composition and pH to create a discontinuous voltage and pH gradient

• the different buffer zones are stabilized by at least 2 different gel layers

what is the “stacking gel”

• the upper layer

• through which the first sample passes

• it is a large pore gel

• non-restrictive to the protein sample

• usually Tris-HCl at pH 6.8

pH of running gel

Tris-HCl at pH 8.8

pH of electrode buffer

Tris-Gly at pH 8.3

what kind of buffer is the stacking gel made in?

what about the tank buffer?

• contains an ion (usually an anion) whose electrophoretic mobility is greater than that of the protein

• the tank buffer or electrode buffer must contain an ion whose mobility is less than that of the protein

explain what happens when electrophoresis begins

• as electrophoresis begins the “leading ion” in the stacking gel moves faster than the protein and leaves behind it in a zone of lower conductivity

• the higher voltage gradient of the zone causes the protein to move faster and to “stack” at the boundary between leading and terminating zones

what kind of gel is below the stacking gel?

• deeper gel with a smaller pore size

• known as resolving or separating gel

• gel is prepared in a buffer of high concentration and pH (in an anionic system)

• in this environment the mobility of the terminating ion increases so that its boundary moves ahead of the protein

SDS-PAGE with discontinuous buffers

• discontinuous system for resolving proteins denatured with SDS

• the leading ion in the Laemmli buffer system is chloride and the terminating ion is glycine

• the resolving gel and stacking gel are made up in Tris-HCl buffers (of different concentration and pH), while the tank buffer is Tris-glycine

• all buffers contain 0.1% SDS

detection of proteins in the polyacrylamide gel

• once proteins have been subjected to electrophoresis

• proteins can be visualized directly by using protein-specific stains

• alternatively, proteins can be transferred to a surface matrix like nitrocellulose or PDVF where proteins can be identified using radioactive or affinity labels or specific Abs

coosmassie blue binding

• in acidic conditions?

stain binds hydrophobically to the backbone of the protein molecules

• therefore binding is nearly linear in binding to different proteins

• acidic conditions, the dye binds to proteins primarily through basic amino
  acids (primarily arginine, lysine and histidine), and the number of coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein.”

silver stain binding

• the silver ion binds to the charged side chains of amino acids contained in the proteins

• therefore the silver stain produces various shades of blue, red, yellow and black depending on the relative abundance of each various aa with ionic side chains

what does electrophoretic transfer allow

• binding of macromolecules to a surface matrix increases the sensitivity of detection methods like autoradiography

• it permits detection of specific Abs or affinity labels and of specific nucleic acids by hybridization with complementary strands of RNA or DNA

can results from electrophoretic transfer be quantitative or qualitative?

• qualitative mostly

• usually do not result in quantitative transfer because of complex nature of the reaction

what type of gel allows for a more efficient transfer SDS or acid/urea gel?

• acid/urea gel

what does the transfer depend on?

• nature of the sample

• kind of binding paper

• pore size of the gel

• buffer used for transfer

monoclonal antibodies

• generation

• describe their affinity/what they recognize

• are generated by identical B cells which are clones from a single
  parent cell.

• This means that the monoclonal antibodies have monovalent affinity and only
  recognize the same epitope (antibody binding site) of an antigen (a substance that stimulates immune response).

polyclonal antibodies

• generation

• describe their affinity/what they recognize

• pAbs) are mixture of heterogeneous which are usually produced by
  different B cell clones in the body. They can recognize and bind to many different epitopes of a single antigen.

• Polyclonal antibodies are produced by injecting an immunogen into an
  animal

detection of Beta-galactosidase protein on PDVF filter

• use an anti-Beta-galactosidase mouse monoclonal antibody

• once the Ab has binds the protein the complex will bind an anti-mouse IgG alkaline-phosphatase conjugate

• this complex is an antibody which recognizes the mouse IgG and has an alkaline phosphatase enzyme covalently linked to it

• this protein-antibody-antibody-AP complex will react with BCIP (substrate of AP) in conjunction with NBT for colour detection (purple/grey colour) of AP activity

how is the previous detection system different from other stains?

• coomassie blue and silver stain detect most proteins, but this method is specific to Beta-galactosidase protein