Protein concentration calculation

Protein Assay by Absorbance

Absorbance assays are fast and convenient, since no additional reagents or incubations are required.

No protein standard need be prepared.  Any non-protein component of the

solution that absorbs ultraviolet light will interfere with the assay. These can be nucleic acids, lipids.

Beer Lambert Law: A= l x c x

A= -log10 I/Io

C= Analyte concentration ; = absorption coefficient wavelength dependent

Principle of the test

Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm.

Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm

Peptide bonds are primarily responsible for the peak at 200 nm

The intensity of the absorbance is proportional to the number of aromatic Amino acids in the protein

BSA: bovine serum Albumin

Absorbance

Protein Absorbance is Max at 280 nm

DNA Absorbance is Max at 260 nm

The DNA/PROT at A280 is higher thanPROT only sample

How to calculate Concentration If nucleic acid are present in the protein sample

they will interfere with the absorbance at 280 nm (A280).

Use the following formula to estimate protein concentration and remove nucleic Acid interference at A260nm.

Protein Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260).

Example: if A280= 1.2 and A260 = 0.3 the PC= (1.55 x 1.2) – (0.75 x 0.3) = 1.635 mg/ml

Serial Dilutions

IntroductionMany of the laboratory procedures

involve the use of dilutions. It is important to understand the

concept of dilutions, since they are used throughout all areas of the clinical laboratory.

Serial Dilutions A serial dilution is any dilution where

the concentration decreases by the same quantity in each successive step.

Serial dilutions are mutiplicative.

What Does This Mean?? If a solution has a 1/10 dilution the

number represents 1 part of the patient sample added to 9 parts of diluent.

So the volumes used would be 10-1= 9. This represents 1 part patient sample

added to 9 parts of diluent.

Doubling Dilutions “Doubling dilutions” are very popular. This is a series of ½ dilutions. Each

successive tube will ½ the amount of the original concentrated solution.

If this is done 6 times this is what you would end up with:

Doubling Dilution 6 Times 1st dilution = 1 /2 2nd dilution = 1 /2 x 1 /2 = 1/4 3rd dilution = 1/4 x 1 /2 = 1/8 4th dilution = 1/8 x 1 /2 = 1/16 5th dilution = 1/16 x 1 /2 - 1/32 6th dilution = 1/32 x 1 /2 = 1/64 This results in a series of dilutions,

each a doubling dilution of the previous one

Dilution FactorThe dilution factor is the final uses

the formula volume/aliquot volume.EXAMPLE: What is the dilution

factor if you add 0.1 mL aliquot of a specimen to 9.9 mL of diluent? The final volume is equal to the aliquot

volume PLUS the diluent volume: 0.1 mL + 9.9 mL = 10 mL

The dilution factor is equal to the final volume divided by the aliquot volume: 10 mL/0.1 mL = 1:100 dilution

Practice Problem: What is the dilution factor

when 0.2 mL is added to 3.8 mL diluent?

Set Up The Problem dilution factor = final volume/aliquot

volume 0.2 +3.8 = 4.0 total volume 4.0/0.2 = 1:20 dilution

Problem Continued Remember that serial dilutions are

always made by taking a set quantity of the initial dilution and adding it successively to tubes with the same volume.

So each successive dilution would be multiplied by the dilution factor.

Problem Continued So in the above problem all successive

tubes would have 3.8 mLs of diluent. You would then transfer 0.2 of the initial

diluted sample into the next tube, mix transfer 0.2, mix and so on.

If you had 4 tubes what would be the final dilution of tube 4?

Solving the Problem – *Calculate Dilution Factor of tube 1

TubeTube 11 22 33 44

AliquotAliquot 0.20.2 0.20.2 0.20.2 0.20.2

DiluenDiluentt

3.8 3.8 3.8 3.8

MathMath *4/0.*4/0.22

1/20x1/201/20x1/20 1/400x1/21/400x1/200

1/8000x1/201/8000x1/20

DilutioDilutionn

1:201:20 1:4001:400 1:80001:8000 1:160,0001:160,000

Solving the Problem Or if you simply wanted to know the

dilution of the final tube you could just multiply them together:

1/20 x 1/20 x 1/20 x 1/20 = 1:160,000

To Measure Immune reactionWe use the Titers TITERS are reported out as the

reciprocal of the last tube giving a positive allergic reaction (ALR).

So if tube 2 gave the lowest ALR, the dilution is 1:800 the titer is reported out as 800/1= 800.