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SDS-PAGE for

Molecular Weight
Determination
Gel Elecrtiphorisis Equipment:
SDS-PAGE for Molecular Weight Determination
To determine the molecular weight of an unknown protein, you should
separate the sample on the same gel with a set of molecular weight
standards.
After running the standards and the unknown protein sample, the gel
is processed with the desired stain and then de-stained for about 12 to
14 hours to visualize the protein bands.

After running the gel, you should then determine the relative
migration distance (Rf) of the protein standards and the unknown
protein. The migration distance can be determined using the following
equation:

Rf=migration distance of the protein


Migration distance of the dye front
SDS-PAGE the result
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SDS-PAGE standard curve
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Plot log molecular mass


versus relative mobility
Molecular mass determination by gel filtration chromatography
Calibrate the column: determine elution volumes of proteins with known
molecular weights--construct a calibration curve relating(known) molecular weight to
(measured) elution volume specifically for that column.
Ve / V0

Elution volume (Ve) is the volume of a solvent required to elute a given solute from the column
after it has first contacted the gel. 6
V0 =avolume
Such ofcurve
calibration solvent space
can then surrounding
be used gel;molecular
to estimate the Typically 35%
weight of an unknown protein .
2- Determination of number of subunits

by two-dimensional electrophores
gradient gel electrophoresis was performed for the first
dimension. Native proteins were dissociated into their
subunits by sodium dodecyl sulphate (SDS) in a gel slice,
then applied to SDS polyacrylamide gel electrophoresis for
the second dimension to determine the M(r) of subunits..

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Determination of the number and relative molecular
mass of subunits in an oligomeric protein by two-
dimensional electrophores

pore gradient gel electrophoresis was performed for the first dimension.
Native proteins were dissociated into their subunits by sodium dodecyl
sulphate (SDS) in a gel slice, then applied to SDS polyacrylamide gel
electrophoresis for the second dimension to determine the M(r) of subunits.
The advantage, accuracy, limitations and application of the method are
discussed.