DPP C2

Kolej Kemahiran Tinggi MARA

Lenggong, Perak

JOB SHEET
DIPLOMA IN BIOCHEMICAL ENGINEERING TECHNOLOGY
PROGRAMME
(FOOD)
SESSION JANUARY – JUNE 2023 SEMESTER 5
EGF 5043
CODE & COURSE SHEET NO JS02
Halal Food Management System
LECTURER DURATION 3 hours

TOPIC Chemical analysis: Protein analysis (Demonstration)

SUB-TOPIC 1) Protein analysis by SDS Page Gel Electrophoresis

TOPIC 1) To detect and differentiate the source of gelatin added in

LEARNING processed foods using of sodium dodecyl sulphate-
OUTCOME polyacrylamide gel electrophoresis (SDS-PAGE)

TOOLS / 1. Protein ABC (sample for testing)

EQUIPMENTS / 2. Electrophoresis Equipment (casting stand, electrophoresis
MATERIALS stand and apparatus)
3. Power supply
4. Protein sample
5. Bio-Rad Laemmli Sample Buffer (contains SDS and either
sucrose or glycerol)
6. 2-Mercaptoethanol (reduces disulfide bonds, disrupts protein
cross-links)
7. MW Markers (already prepared in sample buffer)
8. deionized water
9. Ethylenediaminetetraacetic acid disodium salt (EDTA)
10. Bromophenol blue
11. 8 M urea
12. glycerol
13. 30% acrylamide/Bis (Warning: Acrylamide is a neurotoxin)
14. 1.5 M Tris, pH 8.8
15. 0.5 M Tris, pH 6.8
16. 20% SDS
17. 10% ammonium persulfate
18. TEMED, pH 8.9
19. Coomassie staining/ Silver staining
20. 5, 15, and 50 mL centrifuge tubes
21. 10, 100, and 1000 μL pipette tips
22. 5 and 10 mL pipettes

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23. Filter paper/ Kimwipe

DRAWING AND
DATA

Assemble electrophoresis as shown below :

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Gel Cassette Assembly (Bio-Rad Mini Protean 3)

 Clean and completely dry the glass plates, combs, and any
other pertinent materials.
 Place a short plate on top of a spacer plate. Insert both plates
INSTRUCTION into the green casting frame on a flat surface. Be sure that the
"legs" of the casting frame are down. Clamp the casting frame
and check that the plates are level on the bottom.
 Put the casting frame into the casting stand.

PROCEDURE :
STEP KEY POINT
1. Preparation of the Gel (Warning: Acrylamide is a
neurotoxin. Use gloves, do not
 Combine all reagents except the TEMED for the ingest.)
14% separating gel.

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PROCEDURE :
STEP KEY POINT
note: if Laemmli sample buffer is
14% Separating Gel Components (4.195 not available, you can use 10 mL
mL) 8 M Urea-SDS sample
buffer containing 8 M urea, 2%
-1.184 mL deionized water SDS, 10 mM ETDA, 0.5 M Tris-
-1.96 mL 30% acrylamide/Bis (Warning: HCl, 1.114 g/ml 2-
Acrylamide is a neurotoxin. Use gloves, mercaptoethanol, 10% glycerol,
do not ingest.) 0.05% bromophenol blue.
-1.0 mL 1.5 M Tris, pH 8.8
-21 µL 20% SDS
-12 µL 10% ammonium persulfate
-18 µL TEMED, pH 8.9

 When ready to pour the gel, quickly add the

TEMED, mix using a Pasteur pipette, and transfer
the separating gel solution between the glass
plates in the casting chamber to about 3/4 inch
below the short plate.

 A small layer of butanol can be added on top of

the gel prior to polymerization to straighten the
level of the gel and remove unwanted air bubbles
that may be present. Butanol will not mix with the
aqueous separating gel solution. Once the gel has
polymerized, the butanol can be removed by
absorption with Kimwipes or filter paper. Rinse the
top layer of the gel with dI water prior to pouring
the stacking gel.

 Insert the well forming comb into the opening

between the glass plates.

 Combine all reagents except the TEMED for the

5% stacking gel.

5.1% Stacking Gel (2.484 mL)

-0.4 mL deionized water

-1.8 mL 30% acrylamide/Bis
-0.25 mL 0.5 M Tris, pH 6.8
-12 µL 20% SDS
-17 µL 10% ammonium persulfate
-5 µL TEMED

 When ready to pour the gel, quickly add the

TEMED, mix using a Pasteur pipette, and transfer
the stacking gel solution between the glass plates
in the casting chamber.

 Both the separating and stacking gels should

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PROCEDURE :
STEP KEY POINT
polymerize within six minutes.

 Once the stacking gel has polymerized, the comb

can be gently removed. The polymerized gel
between the short plate and spacer plate forms
the "gel cassette".

2. Sample Preparation

Control samples
 Control samples of pure porcine (PSS) and
bovine (BSS) gelatins were prepared by dissolving
gelatin powder in 10 ml of Laemmli sample buffer
(9.5 mL Laemmli sample buffer plus with 0.5 mL
of 2-mercaptoethanol )
note: if Laemmli sample buffer is not available,
you can use 10 mL 8 M Urea-SDS sample
buffer containing 8 M urea, 2% SDS, 10 mM
ETDA, 0.5 M Tris-HCl, 10.114 g/ml 2-
mercaptoethanol, 10% glycerol, 0.05%
bromophenol blue.
 The mixture was then vortexed until completely
dissolved.
 Subsequently, the mixture was centrifuged at
3000 rpm for 3 min using a centrifugation and 100
μl of the supernatant that contained approximately
80 μg of protein content was collected and loaded
onto the well for electrophoresis procedures.

Processed food (sample testing/sample ABC)

 The confectionery (gummies and marshmallow)
were cut into small pieces by using clean and
sharp scissors and transferred into conical flask
containing deionised water.
 The mixture was stirred on the hot plate until it
completely dissolved.
 This experiment was performed at approximately
37°C

Extraction of gelatin

 Meanwhile, add 50 mL of 2-mercaptoethanol to

950 mL of Laemmli sample buffer.

 Combine 10 mL of each sample (gummies and

marshmallow) with 20 mL of Laemmli sample
buffer plus 2-mercaptoethanol in microcentrifuge
tubes.

 In separate tubes, aliquot 10 mL of MW marker.

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PROCEDURE :
STEP KEY POINT
(MW markers are already prepared in Laemmli
sample buffer.)

 Boil the samples for no more than 5 minutes to

fully denature the proteins. Leave the samples at
room temperature until ready to load onto the gel.

3. Electrophoresis

 Remove the gel cassette from the casting stand

and place it in the electrode assembly with the
short plate on the inside.

 Slide the electrode assembly (with the gel

cassette) into the clamping frame. Press down on
the electrode assembly while clamping the frame
to secure the electrode assembly. This step is
important to minimize potential leakage during the
electrophoresis experiment.

 Pour some 1X electrophoresis buffer into the

opening of the casting frame between the gel
cassettes. Add enough buffer to fill the wells of the
gel. Use a gel loading tip to pipette some buffer
into each well to ensure cleanliness.

 When all wells are sufficiently cleaned, slowly

pipette no more than 30 mL of denatured sample
or MW marker into each well. A yellow guide can
be placed on top of the electrode assembly to aid
in loading the gel.
 When the gel has been loaded, lower the
clamping frame into the electrophoresis tank.

 Fill the region outside of the frame with 1X

electrophoresis buffer.

 Cover the tank with the lid aligning the electrodes

(black or red) appropriately.

 Connect the electrophoresis tank to the power

supply.

 Allow the samples to run at 30 mA until the dye

front reaches the bottom of the gel. This can take
as long as 1 hour.

 When electrophoresis is complete, turn off the

power supply and disassemble the apparatus.

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PROCEDURE :
STEP KEY POINT

4. Staining
 After the gel has been carefully removed, you
may place it in an empty pipette tip box filled with
15 ml of staining solution (Coomassie staining/
silver staining) and 15 ml of 20% acetic acid.
 The gel should stain for 25 minutes while being
gently shaken.
 After this time, the staining solution should be
removed, and the destaining (6:1:3 water:acetic
acid:methanol) solution should be added to
visualize the protein bands on the gel.
 It should also be shaken during this time and the
solution changed periodically to facilitate efficient
gel destaining.

Subsequently, the stained gel was scanned using a

densitometer (GS-800 Calibrated Densitometer Bio-rad,
Hercules, and CA).

RESULT:

Put the printout of a labeled photo of your gel in your result.

QUESTION/DISCUSSION:

1. What is the purpose of SDS-PAGE in this lab exercise?

2. Explain the functions of the following components of the 1X sample treatment buffer
that was added to your samples before loading them onto the electrophoresis gel:
(a) SDS
(b) Mercaptoethanol
(c) Bromophenol blue
(d) glycerol

Describe any difficulties that you had with the sample dilution, the gel loading, and the gel
staining techniques

CONCLUSION:

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