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Sds Page Protocol

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University of Illinois at Urbana Champaign College
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Protocol for performing protein electrophoresis: SDS-PAGE
Purpose
The aim of the experiment is to determine the molecular weight of five unknown
protein samples. In order to so, a sodium dodecyl-sulfate polyacrylamide gel electrophoresis
will need to be performed.
Hypothesis
It is hypothesised that smaller will migrate faster through the polyacrylamide gel, while
larger proteins will migrate slower or lag behind. This will result in varying distances migrated
by proteins, and distance of migration will be directly related to protein size. The proteins will
be visible on gel and appear as blue bands since they will be stained with Coomassie blue. It
will allow us to distinguish the distance each protein has migrated.
Background
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the most
popular form of protein electrophoresis and is commonly used to separate proteins based on
their molecular weight (Nowakowski, Wobig, & Petering, 2014). SDS is an amphipathic
surfactant, it is used in protein electrophoresis as a denaturant (Bio-rad Bulletin 6040 Rev A,
n.d.). SDS binds to and unravels proteins from their tertiary conformities, rendering said
proteins into an elongated rod-like form, masking its intrinsic charge and applying an overall
negative charge to the protein (Bio-rad Bulletin 6040 Rev A, n.d.). In addition, SDS binds at a
consistent rate (1.4g SDS per 1g protein) thus imparting a negative charge which is proportional
to the molecular mass or polypeptide chain length (Nowakowski, Wobig, & Petering, 2014;
Bio-rad Bulletin 6040 Rev A, n.d.). This allows for protein molecular weight estimation since
proteins will migrate, based on size alone, through the polyacrylamide gel.
The polyacrylamide gel is a cross-linked matrix and acts as a sieve through which the
proteins move in response to an electric field (Bio-rad Bulletin 6040 Rev A, n.d.). The rate of
migration and separation is dependent on the acrylamide concentration, where a higher
concentration (higher acrylamide percentage gel) results in slower migration and vice versa
(Good, E., 2021). As in the aforementioned paragraph, proteins will be negatively charged and
will therefore migrate toward the positively charges electrode smaller proteins will migrate
faster and will be found lower down in the gel and larger proteins slower and will be found
higher up in the gel (that is, the higher up the protein in the gel, the larger the protein or
molecular mass). Once the proteins are electrophoresed, Coomassie blue dye is generally used
to stain the polyacrylamide gel for visualisation of the proteins. SDS-PAGE is used for several

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purposes, namely, protein identification and quantification (western blotting), determining
protein purity and estimating protein size (Nowakowski, Wobig, & Petering, 2014).
The aim of this experiment is to determine the molecular weight of five unknown
protein samples by SDS-PAGE. Size will be measured in relation to distance migrated by a
molecular weight marker standard of known molecular weight.
Materials and Methods
Equipment
Bio-rad Mini-PROTEAN® Tetra Vertical Electrophoresis Cell
Bio-rad Mini-PROTEAN® Tetra Vertical Electrophoresis Power Supply
Bio-rad Mini-PROTEAN® Tetra Vertical Electrophoresis Precast gel
Filter paper
Micropipettes (P100, P20, P10)
Microfuge tubes
PCR tubes
Thermocycler
Methods
1. Prepare the following buffers:
Running buffer with SDS (10X):
Mix 30.4g Tris base, 144.2g glycerine, 10g SDS and make up volume to 1L
with distilled water.
Running buffer with SDS (1X):
Mix 100ml of 10X running buffer with 900ml with distilled water.
Sample buffer with SDS (2X):
Mix 0.98g Tris base, 10ml SDS (20%), 0.05g bromophenol blue, 7.98ml
glycerol and make up volume to 50ml with distilled water. Adjust to pH6.8
with concentrated HCl.

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Protocol for performing protein electrophoresis: SDS-PAGE Purpose The aim of the experiment is to determine the molecular weight of five unknown protein samples. In order to so, a sodium dodecyl-sulfate polyacrylamide gel electrophoresis will need to be performed. Hypothesis It is hypothesised that smaller will migrate faster through the polyacrylamide gel, while larger proteins will migrate slower or lag behind. This will result in varying distances migrated by proteins, and distance of migration will be directly related to protein size. The proteins will be visible on gel and appear as blue bands since they will be stained with Coomassie blue. It will allow us to distinguish the distance each protein has migrated. Background Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the most popular form of protein electrophoresis and is commonly used to separate proteins based on their molecular weight (Nowakowski, Wobig, & Petering, 2014). SDS is an amphipathic surfactant, it is used in protein electrophoresis as a denaturant (Bio-rad Bulletin 6040 Rev A, n.d.). SDS binds to and unravels proteins from their tertiary conformities, rendering said proteins into an elongated rod-like form, masking its intrinsic charge and applying an overall negative charge to the protein (Bio-rad Bulletin 6040 Rev A, n.d.). In addition, SDS binds at a consistent rate (1.4g SDS per 1g protein) thus imparting a negative charge which is proportional to the molecular mass or poly ...
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