Experiment 8 – characterization

Materials:

Provided by the students

egg (one for each group)

From the laboratory

1 assay tube with 5 mL of phosphat buffer 0,1mol/L, pH7.8 – Tube 1;

1 assay tube with 3 mL of ninhydrin solution 0,1% in phosphate buffer – Tube 2;

1 assay tube with 3 mL of Biuret– Tube 3;

1 assay tube with 4 mL of distilled water – Tube 4;

1 assay tube with 6 mL of NaCL 1M – Tube 5;

1 assay tube with 5 mL of ammonium sulphate 70% (weight/volume) - Tube 6;

1 empty assay tube – Tube 7;

1 assay tube with 1 mL of phosphate buffer 0,1mol/L - pH7.8 – Tube 8;

1 assay tube with 1 mL of distilled water – Tube 9;

10 mg de Casein (eppendorf)

2 bequers;

3 5ml pippetes ;

HNO₃ 1:1 (at teacher's table);

NaOH 5M (at teacher's table);

Warm-bath.

Metods:

Ninhydrin reaction:

Oppen the egg separating its content in two bequers (albumen in one, egg yolk in another one) without breaching the membrane that involves the egg yolk;

Use the pippete to transfer 1ml of the albumen to the Tube 1.;

Tranfer 2ml of this formed solution to the Tube 2.;

Put the Tube 2 in the warm bath to boil for 5 mim.

Write down the observed results.

Biuret reaction :

TRanfer 1ml of the solution in the Tube 1 (albumen+phosphate buffer) to the Tube 3.

Write down the observed results.

Salting in :

Dilute 2mL of albumen in the Tube 4. Mix it slowly;

Add the NaCl solution in the Tube 4 to it, drop by drop, untill all the material is solubilized.

Write down the observed results.

Salting out:

Adicione solução saturada de sulfato de amônio (Tube 6) ao Tube 4 gota a gota;

Write down the observed results.

Xanthoproteic reaction:

Put 1ml of albumen from the Tube 1 on the Tube 7;

Add 10mg of casein to the Tube 8 and mix it slowly;

Add 5ml of nitric acid (CARE!) to the Tubes 7,8 and 9;

Put the tubes in the warm-bath.

Write down the observed results. Then add 1ml of the NaOH solution, drop by drop (and mix after each drop).

Write down the observed results.

Objectives :

To understand the proteins properties and how they influence the solubilization.

To understand some methods for the identification and quantification of proteins.

To verify the behaviour of differente biomolecules groups in differente solutions.

Report suggestions:

Explain what are the Biuret and Ninhydrin reagentes and how they interact with the proteins.

Explain the major differences among the used methods for solubilizating the proteins and in wich situations each one should be used.

Explçain the mechanisms involved in the solubilization and precipitation of proteins.