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catalyzes a reaction with 4-Chloro-1-napthol (4CN) and peroxide that produces a visible and insoluble purple product…outdated and low sensitivity.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