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PROTEIN SAMPLE (The Important of Protein (involved in manipulation of DNA…
PROTEIN SAMPLE
The Important of Protein
involved in manipulation of DNA and RNA
structural elements (outside and within the cell)
as a receptors ; convey information of extracellular environment
intracellular signaling component that effect the receptor
as a catalyst
key components of the machinery (to determine genes)
Protein Sample Preparation
3) Removal or interfering compounds and changes physio/ chemical properties
4) Protein Quantification
A total amount of protein or concentration of protein can be determined by colorimetric protein assay methods ( Bradford, Lowry, Biuret or BCA Protein Assay
2) Extraction
Protein Extraction Method
2) Resuspend pellet in 5 X sample buffer (SB) by vortexing
3) Boil the suspended cell (95 degree/ 10 min) and centrifuge (max speed/ 1 min
4)Load supernatant that contains total protein of bacteria into well of gel
1) Transfer 1.5 ml of overnight culture into 1.5 ml tube then, centrifuge at max speed and discard supernatant.
1) Cell disruption
by using mechanical or non- mechanical cell disruption
5) Separation via SDS-PAGE
Is a form of electrophoresis that treat samples with SDS to denature proteins
Protein Quantification
Protein are quantified; molecular weights relative to a hydrogen atom.
Range size of protein; 10kD and 220kD (in Daltons)
Protein will migrate according to molecular weights
Molecular weight markers(ladder)
Have been genetically to be specific molecular weights and then bounded to dye molecules so that they are visible on the gel
known as molecular weight
Migration of pattern ladder
Depends on gel type, gel concentration, and running buffer
Protein ladders have coloured reference band for accuracy and convenience molecular weights estimation
Generate a standard curve to calculate protein size
1) Record in a table the distances the five sub 40 kD protein bands of the prestained standard have migrated from the base of the well.
2) Plot the distances migrated (mm) on the x axis agaist the molecular masses (kD) on the y axis
3) Select the data should result in a linear (straight line) curve
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
General Principle of Protein Electrophoresis and SDS PAGE
SDS binds to and coats the protein and keeps them denatured
The net charge of each protein is naturally different (neutral, positive, negative)
Electrophoresis : migration of charged molecules in an electric field
The intrinsic charges of protein are obscured by placing SDS (negatively charge), in both the sample buffer and the gel running buffer
Protein Migration In SDS
1) Protein (negatively charged due to SDS) move to positive electrod.
2) Protein separate by size
3) Smaller proteins move faster
SDS Apparatus
Power Pac
Lid
Glass Plates; short and tall
Casting Stand
Gel Box
Casting Frame and Glass Plate Sandwich
Clamping Frame and Electrode Assembly
Plastic Comb
Electrodes
Loading Buffer Contains
Glycerol, Tris-Cl, SDS, B-Mercaptoethanol, Bromophenol Blue
