Physiology 3140A Study Guide - Quiz Guide: Bovine Serum Albumin, Phosphate-Buffered Saline, Western Blot
22 May 2018
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Western Blot Module
- Used to quantify protein expression
- Steps of western blot:
1. Isolate protein from sample (either tissue or cells)
▪ If the protein of interest is intracellular, use detergent to rupture cells
▪ If protein of interest is extracellular, grind tissue using homogenizer
2. See if protein isolation was equivalent bw samples (Quantify the protein)
▪ Determine how much units of proteins (in mg) in the volume of isolation buffer
(in mL)
3. Load equal concentrations of total protein from each sample in a gel matrix to be
separated by size
4. Transfer separated proteins from gel to membrane (more resilient)
5. Detect protein of interest with a primary antibody
6. Add a secondary antibody (with an enzyme tag) to recognize the primary antibody to
amplify the signal
7. Add a substrate that the enzyme tag of secondary antibody will cleave
▪ The substrate produces a chemiluminescent light that can be visualized by film
or by specialized camera
- STEP 1: Protein Isolation VIDEO:
o Protein isolation must be done on ice
o Remove growth medium (has all factors that help cell grow in culture, including
proteins in the serum)
o The cells are adhered to the tissue culture plastic and remain adhered after all the media
has been removed
o Wash cells with saline solution (ex: PVS or phosphate buffered saline)
▪ Must wash cells to remove any residual serum
▪ Therefore removing any potential contaminants
o Now we can lyse the cells using a lysis buffer which contains detergents
▪ Cells rupture and release proteins to extracellular environment
o Cells are detached from culture plate
▪ Scraping cells from bottom of plate enhancing lysis reaction by increasing
surface area of the cells to the detergent molecules
o Now collect protein sample in Eppendorf tube for western blot
o Keep protein sample on ice to prevent degradation of sample
- STEP 2: Protein quantification:
o This is done to make sure that equal amounts of each protein are loaded in each well in
the blot
o Allows accurate comparison bw diff experimental conditions
o 2 ways to do this:
▪ bicinchoninic acid (BCA) assay
▪ place small amount of protein sample in individual wells with copper
acid solution
▪ also plate a series of a standard protein (bovine serum albumin) of
known concentration with the copper acid solution
▪ copper is reduced from Cu2+ to Cu1+ in the presence of bicinchoninic
acid (produces a purple/blue colored solution in alkaline conditions)
o VIDEO:
▪ Pipette a small volume of protein sample (1-10 uL) in Eppendorf tube
▪ Aliquoted protein sample needs to be diluted with water
▪ Here, you don’t need to keep it on ice bc the diluted samples will only be used to
quantify total protein
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