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Protein Sample Preparation (Important because it serve as (Receptors,…
Protein Sample Preparation
Important because it serve as
Receptors
Intracellular signaling components
Structural element
Key components of the machinery
Catalyst
Manipulation of DNA and RNA
FIrst steps to prepare
1. Cell disruption
methods
Mechanical disruption
Non-mechanical disruption
Second steps to prepare
2. Extraction/protein solubilization
methods
sample/loading buffer
Contain SDS
denature all proteins by breaking down secondary structure and make it negatively charges
Contain B-Mercaptoethanol
break down disulfide bonds in tertiary structure of proteins
Contain Glycerol
increase the density of protein
Contain Bromophenol Blue
use to track the run protein sample on gel
Contain Tris-CL
creates stable environment for proteins
Third steps to prepare
3. Removal of interfering compounds and changes of the physio/chemical properties
Fourth steps to prepare
4. Quantification
A total amount of protein/concentration of protein
determined by
Protein essay
Estimate via UV absorption spectrum at 280nm
Fifth steps to prepare
5. Separation via SDS-PAGE
form electrophoresis that treats samples withs SDS to denature proteins
protein made by unique combination, so it have
positive charge
Negative charge
Neutral charge
Protein quantification
Steps
What is it?
qualified in terms of their molecular weight relative to a hydrogen atom
range size, 10 kD and 220kD
1. Molecular weight markers (ladder)
Known as molecular weight marker
2. Migration patter of ladder
depend on
Gel concentration
Running buffer
Gel type
have coloured reference bonds for convenience and accuracy during molecular estimation
3. Analyzing an interpreting result
4. Generate a standard curve to calculata protein size
data should be in a linear curve
plot graph - the distances migrated (mm) Vs molecular masses of prestained protein bands (kD)