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catalyzes a reaction with 4-Chloro-1-napthol (4CN) and peroxide that produces a visible and insoluble purple product…outdated and low sensitivity.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

Plant Genetic Transformation

The study material for plant transformation techniques along with the techniques for characterization of transgenic lines can be accessed below:

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Online Interactive Lecture Series on Techniques in Plant Molecular Biology: Part V

Lecture V

If you have any queries please feel free to put them down in the comment section. We will get back to you.

Watch Lecture V on Plant Transformation Methods on Youtube

Virtual DNA isolation experiment from the University of Utah

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Online Interactive Lecture Series on Techniques in Plant Molecular Biology: Part IV

Lecture IV

If you have any queries please feel free to put them down in the comment section. We will get back to you.

Watch Lecture IV on Practical Aspects of Gene Cloning on Youtube

Online Interactive Lecture Series on Techniques in Plant Molecular Biology: Part III

Lecture III

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Watch Lecture III on Different Types of Cloning Vectors in Gene Cloning on Youtube