As part of the gel filtration run, you calibrated the elution volume with proteins of known
molecular weight and generated graph A. The HK activity eluted at 60 ml. You subsequently ran
gel electrophoresis of the purified protein in the presence of SDS with and without the reducing
agent, - mercaptoethanol. The gel included proteins of known molecular weight, and an image of
the gel is shown in B. Using information from gel filtration and gel electrophoresis, determine
the native molecular weight, the subunit composition of the intact protein (number and size of
subunits), and the nature of the interactions between subunits in the native protein. Mw Sample
markus A. B. kD 80 b0 5A 40 5.0 10 204040100 vol Cad
Solution
In this question, in absence of beta-ME, the band is appear between 60 KDa to 40 KDa that
means its molecular weight is around ~50 KDa but when reducing agent is used, beta –ME, than
it come near to 30 KDa that mean some of the subunits attach with disulfide bind and that is
covalently linked, therefore, it come down to 30 KDa while using reducing agent.
From this conclude that rest of the units having total molecular weight around
50-30=20 KDa
In reducing gel, in presence of Beta-ME, there is no band of 20 KDa and 10 KDa band is
appeared in molecular weight marker. This indicate that these subunit is made of up below 10
KDa
It could be 5KDa so the it having 20/5= 4 subunits if protein is symmetric and all the subunits
are linked to 30 KDa molecule with disulfide linkage.
if they made up of 4 KDa than 20/4= 5 subunits hence it is asymmetric in nature.
molecular weight and generated graph A. The HK activity eluted at 60 ml. You subsequently ran
gel electrophoresis of the purified protein in the presence of SDS with and without the reducing
agent, - mercaptoethanol. The gel included proteins of known molecular weight, and an image of
the gel is shown in B. Using information from gel filtration and gel electrophoresis, determine
the native molecular weight, the subunit composition of the intact protein (number and size of
subunits), and the nature of the interactions between subunits in the native protein. Mw Sample
markus A. B. kD 80 b0 5A 40 5.0 10 204040100 vol Cad
Solution
In this question, in absence of beta-ME, the band is appear between 60 KDa to 40 KDa that
means its molecular weight is around ~50 KDa but when reducing agent is used, beta –ME, than
it come near to 30 KDa that mean some of the subunits attach with disulfide bind and that is
covalently linked, therefore, it come down to 30 KDa while using reducing agent.
From this conclude that rest of the units having total molecular weight around
50-30=20 KDa
In reducing gel, in presence of Beta-ME, there is no band of 20 KDa and 10 KDa band is
appeared in molecular weight marker. This indicate that these subunit is made of up below 10
KDa
It could be 5KDa so the it having 20/5= 4 subunits if protein is symmetric and all the subunits
are linked to 30 KDa molecule with disulfide linkage.
if they made up of 4 KDa than 20/4= 5 subunits hence it is asymmetric in nature.
