qualitative analysis of product
DESCRIPTION
Qualitative Analysis of Product. Polyacrylamide Gel Electrophoresis. Analysis of Product. Purity of product Different methods have different levels of detection Electrophoresis: Agarose and PAGE Demonstrates: Molecular Weight, Quantity, Purity, and identity. Electrophoresis. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/1.jpg)
Qualitative Analysis of Product
Polyacrylamide Gel Electrophoresis
![Page 2: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/2.jpg)
Analysis of Product
• Purity of product • Different methods have different levels of
detection • Electrophoresis: Agarose and PAGE• Demonstrates: Molecular Weight,
Quantity, Purity, and identity
![Page 3: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/3.jpg)
Electrophoresis
• Horizontal Agarose Gels–Mainly used for DNA analysis– High sensitivity with ethidium bromide
• Vertical Polyacrylamide Gels - Used for Protein analysis - Sensitivity with Coomasie Brilliant blue 50 ng- IEF electrophoresis- Western Blot technique
![Page 4: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/4.jpg)
Electrophoresis and Movement of Molecules
• Molecules can have distinct charges– Positive or Negative –Net charge will cause different movement through
gel
• Molecules can have different shapes– Linear– globular– Alpha helix
+
![Page 5: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/5.jpg)
V = v
Net charge on molecules determines its attraction to + or - electrode
![Page 6: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/6.jpg)
A voltage difference between either side of gel causes
separation of molecules
+
+
++=
![Page 7: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/7.jpg)
P
Polyacrylamide Gel Creates tunnels in gel for molecules to move through
![Page 8: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/8.jpg)
Principles of Electrophoresis
- Ohm’s Law : voltage is proportional to the current flow and inversely proportional to the resistance of the current flow
Voltage = current x resistance
–Using direct current from power supply an electric potential is applied across the gel– This force results in charge movement through a
gel matrix to its opposite charge
![Page 9: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/9.jpg)
What is electrophoresis?
• Forced migration of charged particles in an electric field Fel = Eq q = charge, E= electric field
• Molecules accelerate rapidly and are slowed by frictional forces
• Electrophoretic mobility is determined as:
v = Eq / f f = friction coefficient
• Mobility is intrinsic to the macromolecule and depends on frictional properties, charge
![Page 10: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/10.jpg)
Macromolecular charge
• Macromolecules have a variable net charge that depends on pH
• pH at which net charge is zero = pI
• Electrical shielding of charge occurs when counterions are solvated
V=
V =
![Page 11: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/11.jpg)
Protein in a salt solution
About Charge• Unlike isolated ions, such
as Na + and Cl-, macromolecules have a variable net charge
• Charge depends on pH • Counter ions provide
electrical shielding• These effects can alter
movement of macromolecules
![Page 12: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/12.jpg)
PAGE
• Native : Protein is prepared with little disturbance to its native form– Proteins can aggregate–Movement of samples through the gel can be
inconsistent• SDS : Sodium Dodecyl Sulfate Is a detergent – Protein coated with a negative charge in
proportion to its molecular weight– Denatures and unfolds protein– Added reducing agents (DTT) break disulfide
bonds and tertiary structure
![Page 13: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/13.jpg)
![Page 14: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/14.jpg)
Agarose gels
• Usually used in DNA analysis • Made up of linear polysaccharide mol wt of
12,000 • Basic repeating unit is agarobiose • Gels are prepared at 1% to 3% providing
tunnels for molecules to move through• DNA can be much larger then most proteins
![Page 15: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/15.jpg)
Horizontal Gels
• Gel Box set up frequently used in DNA analysis
![Page 16: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/16.jpg)
Agarose Gel with DNA Bands
• DNA is negatively charged
• Smaller sized DNA moves faster than Larger DNA
• Markers are used to determine relative sizes of DNA pieces
markers
![Page 17: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/17.jpg)
Uses for PAGE
• Separate from other proteins– Proteins separated by size– Isoelectric point
• Determines–Molecular size of protein–Quantifies the amount present– Displays Impurities– Used in western blot assays
![Page 18: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/18.jpg)
Determine Molecular Weight
1. Run standard molecular weight markers on gel2. Run unknown protein on the same gel3. Create a graph of the mol wt versus distance molecule has moved4. Using the distance the unknown has moved
determine the molecular weight from graph
![Page 19: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/19.jpg)
Molecular Weight Markers
Migration of molecular weight of standards are compared to unknown samplewt std vs unknown
![Page 20: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/20.jpg)
Molecular Weight vs Distance
![Page 21: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/21.jpg)
Western Blot Analysis
![Page 22: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/22.jpg)
SDS Effect on Protein Movement
• Sodium Dodecyl Sulfate denatures protein and covers it with negative charges : moves to + end
• Vertical gels are designed so the top of the gel box is attached to the negative power outlet
• The bottom of the gel box is attached to the positive power outlet
• Movement through the PAGE gel is proportional to mass
![Page 23: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/23.jpg)
SDS Polyacrylamide Electrophoresis
![Page 24: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/24.jpg)
Movement of Proteins on an SDS Gel
Stacking of proteins at top of gel at start
Low weight molecular dye
-
+
Distribution of proteins in a charged field
Protein Migration
Highest Molecular Wt. protein
![Page 25: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/25.jpg)
% Polyacrylamide in Gel
• Gels can be made at different concentrations of polyacrylamide
• Example: gels made at 3%,6%,9% and 12% will produce different openings through which the molecule will migrate
• The larger the opening allows large molecules to move through the gel
![Page 26: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/26.jpg)
Vertical Polyacrylamide Gel Electrophoresis
![Page 27: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/27.jpg)
Equipment for Electrophoresis
![Page 28: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/28.jpg)
Procedure in Short
LoadGe Place Buffer
Equip
![Page 29: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/29.jpg)
Electroporhesis of Samples• 1 part Protein Sample: 1 part
Laemmli Buffer are boiled in Eppendorf tube
• Set up SDS-PAGE electrophoresis (or gel) box by SOP
• Place 25ul of boiled sample: loading buffer into gel wells
• Run at 75 mamp for 1-2 hours• Remove, stain with Coomassie
blue and destain with DI water.
![Page 30: Qualitative Analysis of Product](https://reader035.vdocuments.us/reader035/viewer/2022062802/56814577550346895db247e7/html5/thumbnails/30.jpg)
Laemmli Buffer Constituents1 part Protein Sample: 1 part Laemmli Buffer
• BME (beta-mercaptoethanol) and/or DTT (dithiothreitol) are reducing agents that break disulfide bonds causing proteins to go from tertiary to secondary structure.
• SDS (sodium dodecyl sulfate) is a detergent (soap) that breaks delicate hydrogen bonds in the protein causing proteins to go from secondary to primary structure and puts negative charges all over the protein surface.
• Proteins are pulled downwards through the gel to the anode or positive pole proportional to their mass or MW.
• Broomophenol blue is an indicator dye that moves ahead (or in front) of most of the proteins in the samples.
• Glycerol increases the density of the proteins in a sample so that the proteins will fall to the bottom of the well, minimizing their loss.