BIOC2306 genes antibody lec (immunotechniques)

polyclonal vs monoclonal antibodies

population of antibodies that recognise diff epitopes and have diff affinities for the target

single antibody species prod using a clonal cell line (hybridoma)

what is a hybridoma

hybrid cells created by fusing ab producing B cells with myeloma cells (prod monoclonal Abs)

types of Ab light chain

lambda

kappa

types of antibody heavy chain

alpha

delta

epsilon

gamma

mu

what are the first Ab a cell produces

IgM

which Ab type is produced by alternative splicing

IgD

what gene segment(s) do light chains not have

Diversity (D) segments

order that VDJC is joined

Dand J joined first (heavy chain only)

then to V gene segment

then to C (constant) segment

what is the exception to the rule that all cells contain the same DNA

B and T cells

(antigen receptor diversity)

define recombination

rearrangement of genetic material

where are T cells produced

Originate in bone marrow as precursor cells and then migrate to thymus

mature in the thymus

where are B cells produced

originate i the bone marrow as precursor cells then migrate to secondary lympoid organs (lymph nodes and spleen)

mature in secondary lymphoid organs

define central tolerance

immune system process whereby autoreactive T and B lymphocytes are eliminated (thymus for T cells and bone marrow for B cells)

preventing autoreactive cells maturing and entering the periphery

mechanism of central tolerance

negative selection of autoreactive B and T cells that bind with high affinity for self-antigens, cause their apoptosis

prev autoimmune conditions

how to make a hybridoma

immunisation of mouse (vaccinate it with antigen) so raises B cells against that antigen

Isolate those B cells, fuse them with a tumour cell (myeloma)

screen hybridomas for production of desired antibody

clone single cells

2 types of antibody labelling

Directly or indirectly

examples of direcly labelling antibodies

radioactively (I 125)

fluorescently with FITC

rhodamine (red)

prons and cons of direct antibody labelling

convenient and simple

but health risk, pot. poor signal and can be costly

examples of indirect labelling of antibodies

= use secondary antibody that recognises Fc of primary Ab

secondary Ab linked to e.g. fluorophore FITC, rhodamine or an enzyme (HRP or alkaline phosphatase)

pros and cons of indirect antibody labelling

can amplify the signal - multile 2ndary Ab bind 1 primary or 2ndary carry multiple reporter mols - therefore very sensitive

cost effective, same secondary can be used with multiple primary Abs as recognise same constant region

put have to perform two rounds of detection in protocol, plus wash, etc.

what are immuno dot blots

for protein detection

get protein extracts from diff tissues, immobilise them on nitrocellulose membrane

incubate with Ab

wash

Visualise Ab by testing for conjugated enzyme

See what tissue the protein is/isnt expressed in

western Blot procedure

Extract protein, denature in SDS buffer

electrophorese (PAGE)

transfer to nitrocellulose (electrotransfer)

INcubate with specific Ab

Visualise Ab with 2ndary if need (normally use 2ndary conjgated with HRP)

(detect enzyme activity with HRP)

what reaction does HRP catalyse (horse radish peroxidase)

catalyse the release of light (@425 nm) (chemiluminesence)

by at oxidation of luminol by H2O2

detect spectrophotometrically

define chemiluminescence

emission of light due to chemical reaction

what is immunoprecipitation

use specific Abs to precipitate/isolate specific proteins from a complex mixture

antibodies conjugated to beads (cov or magnetically)

bind proteins in sample, wash away unbound, elute from beads

how are antibodies covalently linked to agarose, magnetic beads or solid supports in immunoprecipitation

Protein A or G bind the Fc region of IgG (monoclonal or polyclonal)

can link proteins A and G to agarose, beads or solid supports

so can immobilise specific proteins

steps of immunoprecipitation

add antibody to cell lysate

add protein A or G

spin to precipitate and remove supernant

Elute the protein from beads/proteinA/G e.g. with SDS

use of immunoprecipitation

look at proteins exp at low level (hard to isolate)

identify PTMs (use Ab specific to PTMs)

identify proteins in complex (Ab to one protein can pull down the entire complex)

Therefore identify if X interacts with Y

how can post translational modifications be identified in immunoprecipiation

can use Ab to pull down protein, specific to modification e.g phosphorylation mark

so pull down all phosphorylated proteins

or can do make Ab specific to that modified protein, so can pull down just that

or pull down a protein, then western blot using Ab specific for PTM form

what is co-immunoprecipitation

identification of proteins in complexex

determine if proteins interact

Ab for one of the proteins

(estern blot with Ab against second protein you want to det. if in complex)

3 methods to detect proteins using antibodies

immuno dot blot

western blot analysis

immunoprecipitation

what is chromatin immunoprecipitation (ChIP)

determines the DNA sequences that are bound by specific proteins or modified histones

use Ab against - TF, chromatin assoc protein or modified histone

method of Chromatin immunoprecipitation (ChIP)

use crosslink DNA and proteins/histones

digest chromatin

immunoprecipitate crosslinked DNA (with Ab)

revrese crosslinking and purify DNA

PCR to amplify target sequences or detect via hybridisation (visualise on gel)

what primers do you use in PCR amplification during chromatin immunoprecipitation

primers against specific gene in PCR reaction to determine if that region is bound byprotein of interest

differernt ways ChIP products can be analysed

PCR: does the protein bind that seq

qPCR: quantify level of protein binding

microarray: determine if protein binds to a large number of diff sequences and its relative affinity

chIP Seq: determine all sequences bound by protein

decribe how a DNA microarray works

have many wells that each contain a DNA probe for a diff sequence, therefore when sample added to each well, can detect what sequences are present (they hybridise with the probe)

how are Cy5 and Cy3 used in DNA microarrays

Cy3 (green) used to label a sample of DNA (before ChIP), whereas cy5 (red) used to label the ChIP enriched-fragments.

the DNA probes are immobilised in each well.

Get green if no comp seq in ChIP sample, yellow if present in both. Get no colour if DNA probe binds nothing. Red if only largely detected in ChIP sample

compare relative levels

what is immunocytochemistry

use Antibodies to detect and visualise specific proteins/antigens in a cell. (det. localisation and id presence) det if expressed

immunocytochemistry vs immunohistochemistry

cytochemistry used on cells

histochemistry used on tissue

what does fixing cells with formaldehyde mean

using formaldehyde to chemically crosslink proteins and other mols, prev further reactions in a cell, inactivates enzymes that could degrade sample

method of immunocytochemistry/histochemistry

fix cells with formaldehyde (cross link proteins and preserve structures)

permeabilise cells e.g. with TritonX-100 (detergent)

allows antibody to enter, then visualise with secondary antibody

how to measure reate of synth/degredation of protein

incubate cells with radiolabelled Met (S35 met) for set time (pulse)

incubate with norm met (chase)

make protein extracts at several time points

immunoppt. with Ab, run on SDS PAGE, quantify S35 met label in protein

wwhat does pulse-chase experiments of GATA-1 show

that modified forms of GATA-1 are degraded at different rates