2. Polyacrylamide Gel Electrophoresis (PAGE) of Proteins
PhD. Dhurata Feta
PhD. Artiona Laze
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This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

3. NETCHEM Remote Access Laboratory Guide
Evaluation of wheat genotypes by polyacrilamide gel electrophoresis
In this laboratory work, you will:
Understand the principle of SDS-PAGE
To become familiar with SDS-PAGE setup
Determine of protein molecular weight
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This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

4. Background
Wheat is one of the most important cereal crops worldwide in terms of production and utilization. The ability of wheat flour to be processed into different food is largely determined by the proteins. Mature wheat grain contain 8% to 20% protein.
Wheat is unique among the edible grain because wheat flour has the protein complex called “gluten”. The gluten proteins consist of monomeric gliadins and polymeric glutenins. Glutenins and gliadins are recognized as the major wheat storage proteins.
Glutenins can be broadly classified into two groups, the high molecular weigh (HMW) and low molecular weigh (LMW) subunits, with molecular weigh (Mw) range of 100 to 140 KDa and 30 to 55KDa respectively, according to mobility on SDS-PAGE.
Sodium Dodecyl Sulfate-Polyacrylamide gel Electrophoresis (SDS-PAGE), is a method where charged molecules in solution, mainly proteins, migrate in response to an electrical field.
-This method separates proteins based primarily on their molecular weights.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

5. Aparatus Centrifuge and centrifuge tubes.
Analytical balance - capable of accurate weighing's to 0.01 g.
Heating source (e.g., hot plate)
Vortex (Mixer)
mortar and pestle
mill
Gel casting apparatus
Graduated cylinder or equivalent volume measuring device.
Volumetric Flasks – 50 mL, 100 mL.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

6. Procedure: Protein Extraction Extraction buffer (SDS-based extraction)
TRIS 1M, pH 6.8
Water
SDS
2-mercaptoethanol/heat
Bromphenol blue 1%.
Centrifuge extract
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

7. Procedure: Gel Preparation Set up gel casting apparatus
Mixing of gel ingredients
(follow recipe/directions and adjust to correct pH)
Pouring the gel
Separating gel
Stacking gel
Comb placement/removal
Polymerization
glass sandwich
spacers
cams
Casting stands
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

8. Procedure Buffer Solution for Electrophoresis running
It contains
-TRIS
-Glycine
-SDS
-Water
Staining Solution of Proteins in Gel
It contains:
-Coomassie Blue R 250
-ethanol 95%
- acid trichloroacetic 12%
Gel Decoloration Solution
- Glacial acetic acid
- Methanol
Running Gel, (10% SDS)
It contains:
-Tris-HCl. pH 8.8
-acrylamide
-bis-acrylamide
-SDS 10%
-ammonium persulphat
-TEMED.
Stacking Gel (3% SDS):
-bis-acrylamyde
-TRIS- HCl pH6.8
Water
Ammonium persulphat
TEMED
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

9. The purpose of the stacking gel is to condense the protein into a tight band before running it through the separating gel. This allows better resolution in the separating gel.
The stacking effect works because the stacking gel is made up of a lower concentration of polyacrylamide than the separating gel.
The protein stacks because the proteins move very quickly through the stacking gel, only to run into the separating gel
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

10. Inserting the comb to form wells
Procedure:
Pouring the gel
Inserting the comb to form wells
Removing the comb
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

11. Procedure: Loading the gel
1. Fill wells with distilled water or tank buffer as a rinse
2. Pour off rinse and ½ fill with tank buffer
3. Place extract into sample well using a digital pipette with gel loading tips. Sample size loaded will vary on species and gel type.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

12. Determine Molecular Weight
1. Run standard molecular weight markers on gel
2. Run unknown protein on the same gel
3. Create a graph of the mol wt versus distance molecule has moved
4. Using the distance the unknown has moved determine the molecular weight from graph
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

13. Molecular Weight Markers
Migration of molecular weight of standards are compared to unknown sample.wt std vs unknown
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

14. Molecular weight estimation by SDS-PAGE
Calibration curve for molecular weight estimation.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

15. DESCRIPTION OF THE EDUCATIONAL ELEMENT
Educational element title
Evaluation of wheat genotypes by polyacrilamide gel electrophoresis
Educational field
Chemistry
Level of study
Master academic studies
Title of course in which Educational element is implemented (lecture or lab exercise)
Food Chemical Analysis
Title of teaching unit
Polyacrylamide Gel Electrophoresis (PAGE) of Proteins
Teacher
PhD Dhurata Feta, PhD Artiona Laze
Target group (study program, year of study)
Master Study Programme –Food Analysis, 1st year
Educational objectives of educational element
The chemist show the principle of SDS-PAGE and how to determine protein molecular weight
Required preliminary knowledge and skills
Knowledge's of Instrumental Methods of Analyses
Material available at Moodle platform for the educational element:
Type (.mp4/.avi/.ppt/.pdf/.doc/.jpeg …):
Size (MB):
Used language in the material:
Video clip (.mp4)
16 MB
english
Remote access classroom-laboratory No
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

16. DESCRIPTION OF REMOTE ACCESS NETCHEM COMMUNICATION SIDES
(NOTE: NETCHEM Communication is defined as event that involves all kinds of internet interactions (in real time and not in real time) between participants via devices (PCs, laptops, tablets andmobilephones))
host side
(NOTE: Host side of NETCHEM Communication is defined as PC who invites other users to join the session)
guest side
(NOTE: Guest side of NETCHEM Communication is defined as PC who joins the invitation to session)
COMMUNICATION SOFWARE
Team Viewer
Meeting:
Remote control:
Meeting and Remote control simultaneously:
Skype
Call 1:1:
Conference Call:
COMUNICATION HARDWARE
on host side
on guest side
INFORMATION EXCHANGE TYPE
Educational (one side is dominantly receptive)
Place of Educator participant:
Number of educator(s):
Place of student participant:
Number of student participant(s):
Consultative (two sides are equal in giving-receiving information)
Number of host side participant(s):
Number of guest side participant(s):
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

17. Author, Editor and Referee References
This remote access laboratory was created thanks to work done primarily at University of Niš.
Contributors to this material were: PhD. Dhurata Feta, PhD. Artiona Laze, full professors
Refereeing of this material was done by: _____________________
Editing into NETCHEM Format and onto NETCHEM platform was completed by: ______________
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.

18. References and Supplemental Material
The NETCHEM platform was established at the University of Nis in through the Erasmus Programme. Please contact a NETCHEM representatives at your institution or visit our website for an expanded contact list. The work included had been led by the NETCHEM staff at your institution.
______________________________________________________________________________________________________
This project has been funded with support from the European Commission. This publication reflects the views only of the authors, and the Commission cannot be held responsible for any use which may be made of the information contained therein.