Laboratory session 1:

Review of safety rules and basic laboratory procedures.

Introduction:
Biotechnology experiments involve procedures and the use of reagents and equipment that may
pose a hazard if the proper safety guidelines are not followed. We will be discussing safety
precautions and procedures that apply in a biotechnology setting and review general biological and
chemical safety guidelines, including storing and handling hazardous materials. Since specific
safety precautions are required for conducting each particular experiment, potential safety risks
will be addressed prior to initiate individual laboratory practices. In this first laboratory session we
will review potential hazards and safety procedures for performing separation and analysis of
proteins by electrophoresis.
Chemical safety: Precaution have to be taken when working with chemicals used in all
procedures. Students must review the manufacturers safety data sheet (MSDS) detailing the
properties and precautions for all chemicals utilized as well as the safety sheets prior to starting
the procedures. General handling procedures include using protective gloves for all protocols and
weighing hazardous materials in a hood while wearing a disposable dust mask.
MSDS (Material Safety Data Sheets): a Material Safety Data Sheet (MSDS) is a document that
contains information on the potential hazards (health, fire, reactivity and environmental) and how
to work safely with the chemical product. It is an essential starting point for the development of a
complete health and safety program. It also contains information on the use, storage, handling and
emergency procedures all related to the hazards of the material. The MSDS contains much more
information about the material than the label. MSDSs are prepared by the supplier or manufacturer
of the material. It is intended to tell what the hazards of the product are, how to use the product
safely, what to expect if the recommendations are not followed, what to do if accidents occur, how
to recognize symptoms of overexposure, and what to do if such incidents occur.
Electrical safety: The equipment for performing electrophoresis requires the use of high voltage,
therefore the following precautions have to be taken prior to initiating any experiment. Work area:
should be kept clear of any unnecessary material and the bench and oor should be dry. High-
voltage connections: the high-voltage leads should be intact and not frayed. Plugs should have
protective plastic sleeves. Stackable leads that connect more than one gel unit to a single outlet are
not recommended and should be replaced with shielded-style plugs. A demonstration of proper
handling techniques and use of the equipment will be given by the instructor prior to initiate
the experiment.

Preparation of stock solutions for polyacrylamide gel electrophoresis.

The following stock solutions will be prepared for conducting electrophoretic separation and
analysis of a mixture of proteins in the next laboratory session: Note: gloves should be worn,
while preparing stock solutions for polyacrylamide gel electrophoresis (PAGE); special attention
should be paid while handling acrylamide (since it is a neurotoxin). Mercaptoethanol is irritant
and is readily absorbed through the skin; use in a fume hood; SDS in solid form is a respiratory
irritant, wear a mask while weighing it out.

Stock, buffers solutions Preparation Function

Stock solution 1 Dissolve 30 grams of polyacrylamide Preparation of separating

30% polyacrylamide/0.8% and 0.8 grams of bis-acrylamide in 50 and stacking matrix gels
bis-acrylamide. ml distilled water.
Transfer to a volumetric cylinder and
complete the volume to 100 ml
distilled water.
Filter the stock solution through
Whatman filter paper and store at
4C.
Already prepared stock solution might be
available in the lab.

Pour 80mL Milli-Q water into a 250- Buffer used to prepare

Stock solution 2 ml beaker containing a magnetic separating gel
1.5 M Tris-HCl, pH 8.8 stirring bar, and add slowly 18.2
grams of Tris base while stirring.
When all Tris is completely dissolved,
place a washed pH meter probe in the
solution (the pH will be
approximately 10.3).
Carefully begin adding concentrated
HCl dropwise to the stirring solution.
When the pH reaches the 9.0-9.1
range, let stir for 5 min.
Dilute HCl by adding approximately 1
ml of Milli-Q water to 1 ml of HCl.
Add diluted HCl dropwise to solution
until the pH is 8.8. Let stir 5 minutes
to ensure the pH reading is stable.
Pour solution into graduated cylinder
and add enough Milli-Q water to
bring the total volume to 100 ml.

Preparation of this solution is carried out

similarly to preparation of 1.5 M Tris- Buffer used to prepare
Stock solution 3 HCl, pH 8.8. stacking gel
0.5 M Tris-HCl, pH 6.8 Dissolve 6.0 grams of Tris base
powder in 50 ml of Milli-Q water
Add concentrated HCl slowly,
monitoring pH while stirring.

Sodium dodecyl sulfate
solution (10 % SDS)
Ammonium persulfate
solution (10 % APS)
Tank buffer (electrode
buffer) 0.25M Tris, 1.92M
glycine, 1% SDS, pH 8.5
Loading sample buffer (2X)
4% SDS, 20% Glycerol,
0.12M Tris pH 6.8, 2 %
BPB, 10% BME
1M Tris-HCl, pH 6.8
Staining solution
0.1 % Commassie blue, 10 %
acetic acid, 50 % methanol.
Distaining solution
50 % methanol, 10 % acetic
acid.
After adjusting the pH to 6.8 complete
to 100ml with distilled water using a
volumetric cylinder.
Using a 50-ml beaker, dissolve 1.0 gram Detergent used to denature
SDS in 10 ml of distilled water. proteins
Using a Falcon tube, dissolve 1.0 gram of Chemical used with
ammonium persulphate in 10 ml of TEMED to catalyze the
distilled water. polymerization of
acrylamide and
bisacrylamide
Using a 1 L beaker, dissolve 3.0 g of Tris, Running buffer, establish
14.4.g of glycine, and 1.0 g of SDS in 500 the electric current in the
ml distilled water and complete 1000 ml medium
using a volumetric cylinder (pH should be
8.3-8.5)
Preparation and loading of
Using a 10 ml Falcon tube, mix 0.90 ml of protein samples; SDS
10% SDS, 0.45 ml 100% Glycerol, 0.27 contained in the sample
ml 1M Tris, pH 6.8, 0.63 ml H2O, 0.05 g buffer is used to denature
of Bromophenol Blue, 0.25 ml of - proteins and make them
Mercaptoethanol and vortex. negatively charged.
Dissolve 18.2 g of Tris base with 70 ml
of distilled water; adjust pH with Preparation of loading
concentrated HCl. Add distilled water to a sample buffer
final volume of 100 ml.
Dissolve 0.5 grams of Commassie R-250
brilliant blue in 250 ml methanol, add 50
ml glacial acetic acid and complete to 500 Staining of proteins
ml with distilled water. Mixed for 3-4
hours, filter and Keep at room
temperature.
Mix 250 ml methanol, 50 ml acetic acid Removal of dye from gel,
and 200 ml distilled water. gel distaining
 Laboratory session 2
Polyacrylamide Gel Electrophoresis
Introduction
Principle: SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is one of the most widely used
biochemical method for the separation of proteins based on size. This technique is based upon the
principle that charged molecules migrate in an electric field; protein molecules are denatured with
sodium dodecyl sulfate (SDS) so that they lost their secondary, tertiary or quaternary structure and
acquire uniform charge, therefore the electrophoretic mobility depends primarily on size.

Description: In this laboratory session we will review the setting up of a gel electrophoresis
unit and the preparation and cast of separating (resolving) and stacking polyacrylamide gels. A
bacterial protein extract will be prepared and analyzed by SDS-PAGE.
Please se view these videos before you start:
https://www.youtube.com/watch?v=EDi_n_0NiF4
https://www.youtube.com/watch?v=XUjLO-ek2C8

Materials and Instrumentation:

Reference for assembling the electrophoresis apparatus: Mini-PROTEAN Instruction Manual
(Catalog Numbers 165-8000165-8001)

Assembling the Mini-PROTEAN casting stand and frame

Casting the Gel.
Note: Gloves should be worn, while performing SDS-PAGE. Reminder: special attention should
be paid while using acrylamide (since it is a neurotoxin); to ensure proper alignment, all the
requirements should be clean and rinsed with alcohol. Prepare the separating and stacking gels as
shown in table below.

SDS-PAGE: Preparation of separating (12 %) and stacking (5%) gels

12 % 5%

Reagents Separating gel Stacking gel

Distilled water 3.3 ml 3.7 ml

Stock solution (1) 4.0 ml 0.87 ml

(30% polyacrylamide/0.8%
bis-acrylamide)

Stock solution (2) 2.5 ml .

1.5 M Tris-HCl, pH 8.8

.. 0.67ml
Stock solution (3)
0.5 M Tris-HCl, pH 6.8

10% SDS 100 l 33 l

10% APS* 100 l 33 l

TEMED 5.0 l 5.0 l

Volume 10 ml 5.31 ml

Method: using a micropipette [p1000 (l)- for adding water and stock solutions; p100 or
p200(l)- for 10% SDS and 10% APS and p10(l)- for TEMED], add each solution carefully
into a 25 ml Erlenmeyer flask, swirl to mix after addition of each component and immediately
load gel mixture into the casing.

After pouring the resolving gel solution add EtOH or water on top of gel. Save any leftover
mixture to help you determine when the gel is set. It should take about 30 minutes to polymerize
at room temperature. Remove EtOH or water with a filter paper and add the stacking gel mixture.
Carefully place in the comb avoiding trapping bubbles. After the gel solidifies assemble the Cell
Electrophoresis Module, fill the inner chamber with running buffer and remove the comb.
Sample preparation (protein extracts from E. coli culture): Transfer 3 aliquots (A, B and C) of
1.5 ml O/N of E. coli culture (LB broth) into centrifuge tubes. Spin down at 5000 rpm for 5 mins
and re-suspend the pellet in 75 l loading buffer. Boil for 10 min and spin for 10 min. Pipette
from the very top of supernatant 15 l (A), 10 l (B) and 5 l (C) and load into the gel.

Mini-PROTEAN Tetra Tank Assembly:

Place the Lid on the Mini-PROTEAN Tetra Tank. Make sure to align the color-coded banana
plugs and jacks. The correct orientation is made by matching the jacks on the lid with the banana
plugs on the electrode assembly. A stop on the lid prevents incorrect orientation. Note that the
raised tabs on each side of the tank will now slide through the slots in the lid, guiding the lid to a
proper close. At this point, firmly, yet gently, press down on the lid with your thumbs using even
pressure, till the lid is securely and tightly positioned on the tank.
Power Conditions
1. Insert the electrical leads into a suitable power supply with the proper polarity.
2. Apply power to the Mini-PROTEAN Tetra cell and begin electrophoresis; 200 V constant is
recommended for SDS-PAGE and most native gel applications. The same voltage (200 V) is
used for both 2 and 4 gels. The optimal voltage for your application may differ. Run time is
approximately 35 minutes* at 200 V for SDS-PAGE.
* Electrophoresis time will vary between 35 and 45 minutes for Tris-HCl gels, depending on acrylamide
percentage levels.
Staining and de-staining procedure.
1. Remove SDS-PAGE gel from glass and rinse once in ddH2O in a suitable container with a
lid.
2. Add enough Staining solution to cover the gel and stain for at least 1 hour.
3. Pour off the staining solution (the Coomassie Stain can be recycled a couple of times by
filtering it) and rinse with destain solution.
4. Add fresh Destain solution to cover the gel, destain for 1 hour and replace with fresh solution.
Stop whenever the level of destaining is sufficient (you should be able to see protein bands
without colour background).
5. The used Destain solution can also be recycled by storing it in a sealed container with sponges
or Kimwipes to remove all traces of Coomassie blue Stain.

Visualization and analysis.

Molecular weight standards (track 1 in figure below) are used to determine the migration of
proteins of differing sizes on the gel; the migration is inversely proportional to the molecular
weight and the staining intensity to the protein concentration.
The electrophoretic separation patterns of total protein extracts obtained from an E.coli culture,
as described in sample preparation, are shown in tracks 2, 3 and 4.

1 2 3 4

STD A B C