PROTEIN (Function (Involved in manipulation of DNA and RNA (DNA…
Convey information about the extracellular environment to the cell.
Intracellular signaling components
Within and outside the cell.
Key components of the machinery
Determines which genes are expressed and whether mRNAs are translated into proteins.
Maintain metabolic process
Involved in manipulation of DNA and RNA
RNA splicing or editing.
Protein Sample Preparation
Contaminant removal, Desalting, Concentration (as nedded)
Buffer exchange (desalting)
Remove interfering substances that can negatively impact SDS- PAGE.
Determine the concentration of protein in a sample by protein assay.
Calorimetric protein assay methods
Bradford protein assay
Lowry protein assay
Biuret protein assay
BCA protein assay
Protein are quantified in terms
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)
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
Have coloured reference bands
For convenience and accuracy
Calculate protein size
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.
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.
Sample or loading buffer
Denature all proteins
Break down disulfide bond in tertiary structure of proteins.
Track the run of protein sample on the gel.
Increases the density of the protein sample making it sinked to load in the well
Create stable environment for the protein.
Separation via SDS-PAGE
Sodium dodecyl sulfate-polucrylamine gel electrophoresis
A form of electrophoresis that treats samples with SDS to denature proteins.
Migration of charged molecules in an electric field towards the electrode of the opposite charge.
Have a unique combination of amino acids
May have positive, negative or neutral cherge
The intrinsic charges of proteins are obscured
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
Non-mechanical cell disruption method
Mechanical cell disruption method