Experiment 6 QUANTITATIVE DETERMINATION OF THE SERUM PROTEIN WITH CELLULOSE ACETATE MEMBRANE ELECTROPHORESIS

OBJECTIVE 1) Comprehend the method of electrophoresis. Master the method of cellulose acetate membrane electrophoresis to separate and identify serum proteins. 2) Determine the percentage values of human serum proteins

PRINCIPLE The isoelectric point (PI) of various kinds of serum proteins are generally lower than 7.0, in alkaline buffer solution (PH 8.6), serum proteins are negatively charged, they will migrate toward anode in electric field. The rate of migration will depend on the strength of their net surface charges、shape and size of the proteins.

Use cellulose acetate film as the supporting medium can separate serum protein by electrophoresis in electric field. Serum protein can be separated as 5 zones, from anode to cathode are albumen（Alb）、α1、α2、β and γ-globulin respectively. Then every zone of serum protein can be seen and their percentages can be estimated by spectrophotometer scan. If meanwhile use the Biuret Assay measure the total serum protein concentration, can calculate the concentration of the different protein.

The following table includes the PI and relative molecular mass of the human serum proteins. Protein PI relative molecular mass Albumin 4.88 69 000 α1- globulin 5.60 200 000 α2- globulin 5.60 300 000 β- globulin 5.12 90 000~15 000 γ- globulin 6.35~7.30 158 000~300 000

albumin 1- 2- - -globulin starting point of sample application A   albumin 1- 2- - -globulin cathode anode schematic diagram of normal human serum CAM electrophoresis

PROCEDURE 1 Pretreatment of the cellulose acetate film: Immerse into the electrophoresis buffer 20min before experiment, take it out and absorb with filter paper. 2 Application of sample: add 3~5μl serum on the rough side of membrane at the site of the linedraw, after the sample permeated into the membrane put the membrane on the electrophoresis device ( rough side adown, the sample side at the cathode), balance 5 min. 3 Electrophoresis: in room temperature, voltage 80v 10min, then voltage 100v 30-45 min.

4 Dyeing: dye in amino black B10 for 3-5 min, shake untimely to dye thoroughly. 5 Poaching：poaching with the rinsing solution repeatedly, until the background is colorless. 6 Eluting：observe and judge the shade of color and width of the bands by naked eye, snip the bands in order, then snip the same area as the band of album at the side of the cathode as a blank, put in the marked tubes respectively. Add decolorizer 4ml in the tube of album and blank tube, put 2ml in the rest tubes, place in room temperature for 30min, shake thoroughly every 10min. 7 Determine the absorbance of each tube under 620 nm wavelength.

RESULT and CALCULATION 1 Observe the shade of width and order of the bands and record them. 2 calculate after elution The absorbance of the different constituent of the serum are denoted as Aalbum、Aα1、Aα2、Aβ、Aγ Tube number album α1、 α2、 β γ-globulin Total Absorbance

（1）T=2×A album+ Aα1+ Aα2+ Aβ+ Aγ （2）calculate the percentage content of each electrophoresis zone( different constitute of serum protein) ODα1 2×ODalbum album% α1-% = = T T ODβ ODα2 α2-% β-% = = T T ODγ γ-% = T