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PROTEIN (Function (Involved in manipulation of DNA and RNA (DNA…
PROTEIN
Function
Receptors
Convey information about the extracellular environment to the cell.
Intracellular signaling components
Structural elements
Within and outside the cell.
Key components of the machinery
Determines which genes are expressed and whether mRNAs are translated into proteins.
Catalysts
Maintain metabolic process
Involved in manipulation of DNA and RNA
DNA replication
DNA recombination
RNA splicing or editing.
Protein Sample Preparation
Contaminant removal, Desalting, Concentration (as nedded)
Using
Buffer exchange (desalting)
Protein precipitation.
Function
Remove interfering substances that can negatively impact SDS- PAGE.
Quantitation
Function
Determine the concentration of protein in a sample by protein assay.
Using
Calorimetric protein assay methods
Bradford protein assay
Lowry protein assay
Biuret protein assay
BCA protein assay
Protein are quantified in terms
Molecular weights
Proteins range in size between the range of 10 kD and 220 kD
Relative to a hydrogen atom.
Protein in gel will migrate according to molecular weights.
Molecular weight markers(ladder)
Protein standard
Made by protein which have been genetically engineered to be specific molecular weights
Bound to dye molecules
To visible on the gel or western blot.
Migration pattern of ladder
Dependable on
Gel concentraion
Running buffer.
Gel type
Have coloured reference bands
For convenience and accuracy
Standard curve
Function
Calculate protein size
To create
Measure and record in a table the distances the five sub 40 kD protein bands of the prestained standard.
Measure Kaleidoscope Standard bands between 37 and 10 kD.
Protein solubilization
Funtions
Denature the proteins completely
Disrupt any disulfide bonds through reduction
Dissolve any particles which would interfere with electrophoresis.
For successful electrophoretic separation, proteins must be well solubilized.
Carried out
Sample or loading buffer
SDS
Denature all proteins
B-Mercaptoethanol
Break down disulfide bond in tertiary structure of proteins.
Bromophenol blue
Track the run of protein sample on the gel.
Glycerol
Increases the density of the protein sample making it sinked to load in the well
Tris-Cl
Create stable environment for the protein.
Separation via SDS-PAGE
SDS-PAGE
Sodium dodecyl sulfate-polucrylamine gel electrophoresis
Function
A form of electrophoresis that treats samples with SDS to denature proteins.
Electrophoresis
Migration of charged molecules in an electric field towards the electrode of the opposite charge.
Protein
Have a unique combination of amino acids
May have positive, negative or neutral cherge
The intrinsic charges of proteins are obscured
Strong anionic
Sodium dodecyl sulfate (SDS)
Binds to and coats the protein
Keeps the protein denatures as relatively linear chains.
Protein migrate in a polyacrylamide gel
Mass become main variable affecting the migration rate of ech protein.
Protein have equivalent negative charge densities
Detergent
Cell disruption
Using
Non-mechanical cell disruption method
Mechanical cell disruption method
Function
Release proteins