Vitamins, Harmones and Hemoglobin
Hemoglobin electrophoresis is based on(AIIMS May 2007)
|B||Charge and mobility|
Proteins can be separated based on:
c. Molecular weight
d. Isoelectric PH
Methods based on size:
a. Dialysis: Using dialyzable membrane, proteins of size larger than the pore of this membrane will stay back.
b. Size exclusion chromatography:
Stationary phase is made of polymer beads with pores of particular stokes radius.
Proteins of stokes radius larger than this will tumble down, whereas the other molecules stay in the pores.
Based on charge:
b. Ion exchange chromatography
In protein electrophoresis, we give all the protein molecules a negative charge by using an alkaline buffer, so that they move towards anode on application of current.
(Based on the principle that charged molecules move in an electric field)
The mobility depends upon,
a. Molecular weight
c. Number of charges
Hence proteins gets separated into distinct bands (arranged in the order of farthest to nearest from the point of application)
Since it is of the least molecular weight, it moves farther
Alpha1 Antitrypsin, Retinol binding protein, alfa fetoprotein, and alike molecules of same charge, molecular weight and shape.
Alpha 2 macro globin, Ceruloplasmin
After separation, we quantitate, the amount of protein in each band using a densitometry scanner and
- A thick gamma band is seen in multiple myeloma and chronic infections
b. In the former since it is monoclonal production, they will be of same molecular weight and charge, hence the band will be narrow
c. In the latter since it is polyclonal the band will be broader
- In nephrotic syndrome, since the glomerulus becomes leaky, the smaller proteins are all lost giving rise to a relatively thicker alpha 2 band
- Similarly in haemoglobin electrophoresis, after haemolysing and getting the haemoglobins, using an alkaline buffer, we impart negative charge to them and run the electrophoresis. They get separated based on the differences in their molecular weight. The farthest from the point of application is Hb A, next is Hb F, Hb S, Hb C in that order. So basically haemoglobin electrophoresis is based on charge and mobility and they separate haemoglobins based on molecular weight.
Ion Exchange chromatography:
1. Anion exchange chromatography
2. Cation exchange chromatography
In anion exchange chromatography, we use as stationary phase, synthetic polymer beads carrying positive charge, so that anionic proteins in the protein mixture will get attached to the beads and so proteins with high positive charge will be eluted first. Vice versa happens in cation exchange chromatography.
Based on molecular weight:
Based on Isoelectric PH:
Based on density: