Blotting techniques

are based Complementarity and Hybridization

Blotting techniques are used to answer questions likeo How do we find genes of interest in a genome?  o Or a particular mRNA in the total cellular RNA?   o Or a particular protein out of all cell protein?

The important features of hybridization: • Hybridization reactions are specific - the probes will only bind to targets

with complimentary sequence (or, in the case of antibodies, sites with the correct 3-d shape).

• Hybridization reactions will occur in the presence of large quantities of molecules similar but not identical to the target.

• These properties allow you to use hybridization to perform a molecular search for one DNA molecule, or one RNA molecule, or one protein molecule in a complex mixture containing many similar molecules.

• Factors that affect hybridization characteristics– Strand Length – Base Composition– Chemical environment

Stringency- refers to how likely your probe is to bind to fragments of DNA other than the fragment of interest

• Strand length– The longer the probe the

more stable the duplex• Base Composition

– The % G:C base pairs are more stable than A:T

• Chemical environment– The concentration of Na+ ions

stablize– Chemical denaturants

(formamide or urea) destablize hydrogen bonds.

Denaturation and Tm

The important properties of the three blotting procedures of DNA analysis:

Southern Northern Western

What is separated by molecular weight? (target)

DNA cut with enzymes or amplified

RNA denatured with formamide

Protein denatured with SDS or run as a “native” protein

Probe Labelled Piece of DNA

Labelled Piece of DNA

Labelled Antibody against protein

What do you learn?

Restriction map of probe in DNA or

Whether probe is present in sample DNA

How much of the RNA that the DNA probe codes for is present and how big a piece of RNA is it located in

How much of a particular probe the antibody reacts with is present

How big is the proteinIf native protein –does it

contain a particular protein that the antibody reacts with

The blotting process has the following steps, : - Gel electrophoresis - Transfer to Solid Support - Blocking - Preparing the Probe - Hybridization - Washing - Detection of Probe-Target Hybrids

Gel Electrophoresis This technique separates molecules on the basis of their size or shape.   •Cast slab of gel material, usually  agarose or polyacrylamide.  The gel is a matrix of polymers forming sub-microscopic pores. •The size of the pores can be controlled by varying the chemical composition of the gel. •the distance migrated is roughly proportional to the log of the inverse of the molecular weight The gel is set up for electrophoresis in a tank holding pH buffer.   Electrodes apply an electric field:

PAGE- has smaller pore size- better resolution (2bp differences in DNA) and separates smaller molecules (50-500bp).

• 1 kb piece of DNA = 660kDa• Average protein has mass of ~ 60kDa.• PAGE used for proteins and small pieces of DNA

4 components• Acrylamide-long polymer chains• APS- induces polymerization which generates free radicals (charged oxygens)• TEMED-is a free radical stabilizer (N’N’N’N’-tetra methylene diamine)

, Air inhibits polymerization as it scavenges free radicals• Bisacrylamide- is a cross linking agent and links long polymers of acrylamide

(N, N’-methylene bisacrylamide)

Pore size is determined by % acrylamide and the amount of cross linker

SDS PAGE• SDS is used in the gel mix.• SDS is –ve charged and binds to proteins, it denatures (unfolds)

proteins and gives a net negative charge. • Proteins will then migrate to the anode• Proteins all have same charge to mass ratio• Can be separated based on size aloneNative PAGE• No denaturing agents• Proteins separated based on size charge and shape. • Used when want to keep protein active to study conformation, self-

association or aggregation, and the binding of other proteins

Southern Blot

Transfer: The DNA, RNA, or protein can be transferred to nitrocellulose in one of two ways: 1) Electrophoresis, which takes advantage of the molecules' negative charge:                                                                

2) Capillary blotting, where the molecules are transferred in a flow of buffer from wet filter paper to dry filter paper:

BlockingThe filters are soaked in a blocking solution which contains a high concentration of DNA, RNA, or protein. This coats the filter and prevents the probe from sticking to the filter itself. During hybridization, we want the probe to bind only to the target molecule.

Preparing the probeThe label has to be added to the probe and attached

HybridizationIn all three blots, the labeled probe is added to the blocked filter in buffer and incubated for several hours to allow the probe molecules to find their targets.

WashingAfter hybrids have formed between the probe and target, it is necessary to remove any probe that is on the filter that is not stuck to the target molecules. To do this, the filter is rinsed repeatedly in several changes of buffer to wash off any un-hybridized probe.

Detecting the Probe-Target Hybrids• At this point, you have a sheet of nitrocellulose with

spots of probe bound wherever the probe molecules could form hybrids with their targets. The filter now looks like a blank sheet of paper - you must now detect where the probe has bound.

Usually use1. biotin-strepavidinwith conjugated enzyme or

fluorescence2. RadioactivityNote the membrane is usually blocked with generic protein

e.g. BSA before using one of these protein based visualisations to target probe hybrids to prevent non specific sticking of the visualisation chemicals