Scribd
Upload
39 views2 pages

Module 6

The document outlines steps to isolate, purify, and characterize a theoretical cytoplasmic enzyme from rat liver. The steps include extracting from rat liver using phosphate buffer, subcellular centrifugation to separate particles by size, ammonium sulfate fractionation to precipitate proteins between 35-45% saturation, gel filtration chromatography to separate by size, affinity chromatography using an antibody, SDS-PAGE gel electrophoresis to separate proteins by size, and determining the enzyme's structure using X-ray crystallography.

Uploaded by

Rhena Therese Tordesillas
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
39 views2 pages

Module 6

The document outlines steps to isolate, purify, and characterize a theoretical cytoplasmic enzyme from rat liver. The steps include extracting from rat liver using phosphate buffer, subcellular centrifugation to separate particles by size, ammonium sulfate fractionation to precipitate proteins between 35-45% saturation, gel filtration chromatography to separate by size, affinity chromatography using an antibody, SDS-PAGE gel electrophoresis to separate proteins by size, and determining the enzyme's structure using X-ray crystallography.

Uploaded by

Rhena Therese Tordesillas
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
You are on page 1/ 2

ACTIVITY 6.

PROTEIN ISOLATION, PURIFICATION, AND CHARACTERIZATION

1. Design a protein isolation, purification, and characterization experiment for a theoretical


cytoplasmic enzyme assumed to be a dimeric protein (MW 50 kDA) from rat liver incorporating
the following steps:

a. extraction from rat liver (specify a possible buffer system to use for extraction)

When the Rat Liver is already extracted from the body. Cut it into pieces and place it into a
sterile microcentrifuge tube. Use phosphate buffer to homogenize the tissue to get a
homogenate.

b. sub-cellular centrifugation

The homogenate needs to be centrifuged using Deferential Centrifugation in order for us to


have a high resolution size of sub-microscopic from its microscopic particle.

c. ammonium sulfate fractionation (assume maximum enzyme activity is found in the 35-45%

Assuming that the volume of the homogenate is 10 mL with 40% (NH4) 2SO4. Add 7.04g of
ammonium sulfate to the solution. I got the mass of the ammonium sulfate since 4mL of
ammonium sulfate is required for the 10 mL homogenate and the density of ammonium sulfate
is 1.77g. There are 1.77 grams of ammonium sulfate in every mL of solution. Therefore, 7.04g of
(NH4)2SO4 is needed for the solution. Dialyze the mixture and concentrate the protein. The
denser protein will come out first than the less dense proteins.

d. FPLC with gel filtration (specify a possible solid support and buffer system)

The particles of protein are packed into columns and the sample is added into it. The beads
have pores in them where smaller protein can enter preventing themselves to elute first. As a
result, larger proteins will elute first and after them are the smaller one.

e. FPLC with affinity chromatography

Antibody affinity – identify the antibody to which the protein binds at different pH values using
buffers. Proteins that are not recognized by the antibody will come out first and the recognized
protein will follow after.
f. SDS-PAGE

Samples are added onto a gel of polucrylamide which is placed in an electrophoresis buffer with
electrolytes. Negatively charged molecules will move into the positively charge anode if a
voltage is applied. The small proteins will move out quickly and the while larger proteins will
slowly move out following the direction of smaller protein.

g. Determine the primary structure, secondary, tertiary, and quaternary structure of the
enzyme

In order to determine the primary structure, secondary, tertiary, and quaternary


structure of the enzyme we need to undergo X-ray Crystallography.

2. Draw a schematic diagram of the isolation, purification, and characterization procedure you
have proposed.

Rat Liver Extraction Ammonium Sulfate


Differential Centrifugation
Product: Tissue Fractionation
Product: Homogenate
Product: Proteins

Gel filtration Antibody affinity SDS Page


Product: Enriched Protein Product: Enriched Protein Product: Purified Protein

You might also like