Western blot analysis

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Western Blotting

—  Western blotting is widely used to detect a specific protein in a sample of tissue homogenate or extract.

—  It works on the principle of gel electrophoresis.

—  Proteins are separated based on their size on a polyacrylamide gel.

How we do it!

—  Step I: Isolate protein.

—  Total protein/protein fractions

—  Purified protein/crude protein

—  Step II: Separate protein sample on the basis of MW on PAGE

—  Reducing/Non-reducing gel

—  Step III: Transfer protein from gel to the membrane.

—  PVDF (Polyvinylidene fluoride) membrane used

—  Use of a pre-stained protein ladder helps to detect transfer process

—  Electro-transfer is done. Wet/semi-dry/dry transfer systems are available

—  Step IV: Stain the membrane with ponceau.

—  Checks if the transfer is complete

—  Stain is washed with water before step V.

—  Step V: Blocking is done with skimmed milk to eliminate non-specific antibody binding.

—  Step VI: Antibody probing

—  After blocking, the membrane is incubated with the primary antibody overnight.

—  This is followed by incubation with secondary antibody (tagged with horseradish peroxidase/alkaline phosphatase)

—  The membrane is then washed to remove unbound antibodies

—  Step VII: Detection

—  The membrane is treated with the substrate for HRP/AP which gives chemiluminescence.

—  The signal can be recorded on X-ray film or in a chemi-doc.

Western blot detection

 Colorimetric detection: HRP catalyzes a reaction with 4-Chloro-1-napthol (4CN) and peroxide that produces a visible and insoluble purple product…outdated and low sensitivity.

Chemiluminescence detection: In the presence of HRP and a peroxide buffer, luminol oxidizes and forms an excited state product called 3-aminophthalate that emits light at 450 nm….easy and high sensitivity.

Fluorescence detection: Antibodies are conjugated to a specific fluorophore and can be detected using an imaging system…easy but moderate sensitivity.

Chemiluminescence detection

Alkaline phosphatase-tagged antibody:

·         Enzymatic dephosphorylation of dioxetane substrate by alkaline phosphatase leads to the metastable phenolate anion which, upon decomposition emits light at ≈480 nm.

HRP-tagged antibody:

•      Secondary antibody is tagged with HRP

•      ECL (Enhanced chemiluminesce) substrate contains Luminol, hydrogen peroxide and an enhancer (phenol, naphthol, etc.)

•      In the presence of HRP and a peroxide buffer, luminol oxidizes and forms an excited state product called 3-aminophthalate that emits light at 450 nm

•      Enhance is added so that the reaction can proceed for prolonged durations

•      The reaction emits a light signal at 450 nm

Application of Western Blot

—  Detection of a specific protein in the proteome

—  Detecting phosphorylation states of proteins using specifically designed antibodies. Phosphorylation also makes proteins heavier so that their position on blots gets slightly shifted.

—  Detecting changes in protein levels across treatment groups.

—  Detection of post-translational modification of a protein (i.e. phosphorylation, ubiquitination, etc.) using specific antibodies.

Click here for Western blot animation

Southern and Northern Blotting Analysis

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Blotting technique

—  A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier.

—  The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.

Southern blot

—  Used to study how genes are organized within genomes by mapping restriction sites in and around segments of genomic DNA for which specific probes are available.

—  Combines transfer of electrophoresis -separated DNA fragments to a membrane and subsequent fragment detection by probe hybridization.

—    Named after its inventor, the British biologist Edwin Mellor Southern.

Edwin Mellor Southern

—  The technique is based on HYBRIDIZATION.

—  Hybridization is the process of forming a complementary base-pairing between a single-stranded DNA probe and a single-stranded target DNA.

— The reactions are highly specific i.e. probes will only bind to targets with a complementary sequence.

Principle of hybridization

Transfer methods

—  The transfer of electrophoretically separated DNA from gels to two-dimensional solid supports is a key step in Southern hybridization.

—  Upward Capillary Transfer: DNA fragments carried from the gel in an upward flow of liquid and deposited on the surface of the solid support

—  Downward Capillary Transfer: DNA fragments carried in a downward flow of alkaline buffer and deposited on the surface of a solid support

—  Simultaneous Transfer to Two Membranes: For a high concentration of target DNA, the capillary method can transfer DNA simultaneously to two solid supports

—  Electrophoretic Transfer: Used for analysis of small fragments of DNA separated by PAGE. Only for nylon membranes

—  Vacuum Transfer: DNA and RNA can be transferred rapidly and quantitatively from gels under vacuum

Upward Capillary Transfer

—  The liquid is drawn upward through the gel by capillary action.

—  Rate of transfer depends on:

◦      size of the DNA fragments

◦      concentration of agarose in the gel

—  Small fragments of DNA (<1 kb) are transferred within 1 hour

—  Larger fragments are transferred more slowly and less efficiently.

—  Capillary transfer of DNAs > 15 kb requires at least 18 hours

Set-up for upward capillary transfer

How we do it!

—  STEP-I: Isolate genomic DNA (10 μg per lane required)

—STEP-II: Restriction digestion with one or more enzymes and agarose gel electrophoresis

—  STEP-III: The digested DNA fragments are denatured in the presence of alkali in situ (in gel)

—  STEP-IV: The fragments are then neutralized with NaCl to prevent renaturation before the addition of probe

—  STEP-V: Upward capillary transfer of DNA fragments to the membrane (solid support). Usually, a nitrocellulose or nylon membrane is used.

—  STEP-VI: UV cross-linking of the transferred DNA fragments to the membrane

—  STEP-VII: Preparation of radiolabelled probe usually with α-P³² labelled dCTP or dATP. Fluorescence labeling with Digoxigenin (DIG) is also possible.

—  STEP-VIII: Hybridization of the membrane with the labeled probe under specific conditions. The probe hybridizes with the complementary DNA fragment.

—  Before hybridization blocking is done with salmon sperm DNA to eliminate non-specific probe binding

—  STEP-IX: Wash the unbound probe.

—  STEP-X: Detection by autoradiography

A typical Southern blot

Click here for Southern Blot animation

Application of Southern Blot

·         Primary usage is to identify a specific DNA in a DNA sample.

—  To confirm integration of a transgene in the host genome.

—  To identify copy number of the transgene integrated in the host genome.

Other applications:

—  Identify mutations, deletions, and gene rearrangements.

—  In RFLP

—  Used in the prognosis of cancer and in prenatal diagnosis of genetic diseases.

—  Diagnosis of HIV-1 and infectious disease.

—   In DNA fingerprinting:

◦      Paternity and Maternity Testing

◦      Criminal Identification and Forensics

◦      Personal Identification

Northern Blot Analysis

•     Northern blotting is a technique for the detection of specific RNA sequences in the transcriptome.

•      Northern blotting was developed by James Alwine and George Stark (1979) and was named such by analogy to Southern blotting.

Application of Northern Blot

—  Used for the study of gene expression at the level of mRNA (messenger RNA transcripts).

—  Detection of mRNA transcript size

—  Study RNA degradation

—  Study RNA splicing

—  Study RNA half-life

—  Often used to confirm and check transgene expression.