00. Protein Color Me Try 12 Jan 2012

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Determination of total serumDetermination of total serumproteinsproteins



y include albumin, globulins, fibrinogen and

prothrombin.

y All plasma proteins are synthesized in the

liver except -globulins which are

synthesized in the reticuloendothelial

system.



yNormal Value:

y 6-8 g/dl serum.

y

This value decreases in liver disease (dueto diminished synthesis) and kidney

disease (due to excess protein loss in

urine).



Principle:Principle:

yCopper in alkaline solutions reacts

with peptide bonds of proteins

and polypeptides producing aviolet color.

yThe color intensity is proportional

to protein concentration in themedium.



ReagentsReagents

1- Biuret reagent: A 1 litre of Biuret

reagent contains the following:

NaOH, 0.1N: 4 g/l.K-Na-tartarate (Rochelle salt):

16mmol/l.

Potassium iodide: 15 mmol/l.Cupric sulfate: 6mmol/l.

2- Standard albumin solution: 10 g/dl.



Blank Standard Sample

Distilled water 25 ul - -

Standard - 25 ul -

Serum - - 25 ul

Biuret reagent 1 ml 1 ml 1 ml



y Mix well the contents of the tubes. Leave

them at room temperature for 10

minutes then measure the absorbance of

standard (A standard) and sample (Asample) against blank at wave length 546

nm (540-560 nm) using colorimeter



y Calculations:

y Conc. Sample = A sample/ A Standard

x Conc. Standard «. =g/dl



Determination of serumDetermination of serum

AlbuminAlbumin

y Normal Value:

y 3.5-5.5 g/dl serum.

y

Albumin being a smaller molecule thanthe other fractions of plasma proteins, it

is the major plasma protein fraction to be

excreted in urine in kidney diseases. This

condition is called albuminuria.



P rinciple:

y It is a colorimetric test in which albuminin the presence of bromocresol green at

acidic medium produces a colour

changes of the indicator from yellow-

grean to green-blue.

Formation of an albumin/bromcresol ² green complex at pH 4.2 ² 4.3



P rocedure:P rocedure:

Prepare 3 test tubes labeled blank ,Prepare 3 test tubes labeled blank ,

standard and sample, then pipette in thestandard and sample, then pipette in thetubes according to the following schedule:tubes according to the following schedule:

Blank Standard Sample

Distilled water 10 l - -

Albumin standard - 10 l -

Serum - - 10 l

Bromocresol green 2.5 ml 2.5 ml 2.5 ml



Conc.Of sample =

A sample

X Conc. standard = «g/dL A standard

Mix well and allow to stand for 5 minutes, then read the

absorbance of standard (A standard) and sample (A

Sample) against blank at 630 nm (600-650 nm) using a

colorimeter. The colour is stable for one hour .

Calculation:



Determination of Determination of

Albumin/ Globulin ratioAlbumin/ Globulin ratio(A/G ratio)(A/G ratio)



y Normal value:

A/G = 1.5/1 - 2/1

N.B.y Normal range for serum albumin: 3.5 ² 5.5

g/dl

y Normal range for serum globulin: 2 - 3.6 g/dly Diminished values of A/G ratio indicate

1. liver diseases,

2. kidney diseases, ory Reversed (Diminished) values of A/G

ratio indicate

y

In Multiple Myeloma



y Calculation:

y A=Serum albumin

y G=Serum globulin

N.B.:y Serum globulin = total serum proteins-

serum albumin

Reversed A/G ratio in Multiple myeloma.

00. Protein Color Me Try 12 Jan 2012

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