The document summarizes the purification of lactate dehydrogenase (LDH) from chicken muscle. Key steps included: 1. Homogenizing the muscle tissue to disrupt cells and release LDH. 2. Removing debris via centrifugation. 3. Precipitating and concentrating LDH using ammonium sulfate. 4. Dialyzing the sample to remove salt. 5. Purifying LDH using affinity chromatography on a Cibacron blue column. 6. Analyzing purity via SDS-PAGE gel electrophoresis and activity assays.

2. Purification of lactate
dehydrogenase (LDH) from chicken
muscle
Gaurav Dutta Dwivedi
Hareesha Kakkera

3. Aim of the project
• The purpose of this experiment is
to extract and purify LDH enzyme
from chicken muscle using
appropriate protein purification
techniques.

4. Strategy applied for the
purification process
Techniques applied for achieving this:
• Centrifugation.
• Selective protein precipitation.
• Dialysis.
• Affinity chromatography.
Many different analytical methods were
employed to determine the presence, purity
and concentration of LDH such as activity
assays and SDS-PAGE.

5. The Protein:lactate dehydrogenase
In this course, we used lactate dehydrogenase
(LDH) as the subject of our studies.
It is an vital player in the anaerobic generation of
energy from glucose as well as in the synthesis of
glucose from lactate. It carry out the following
reaction:
Lactate + NAD+ LDH Pyruvate + NADH + H+
Enzyme clears lactic acid from working muscles
The obvious source of enzyme is muscle tissue
(heart & skeletal muscle, H&M, isomers)
We detect in assay for the enzymes ability to
convert Pyruvate to Lactate.

6. Brief overview of the experiment
• Disrupt
– Blender, homoginizer
• Remove debris
– Centrifugation
• Precipitate/concentrate
– Ammonium sulfate
• Remove salt
– Dialysis
• Purify
– Chromatography
• Analyze
– Activity, molecular weight

7. Disrupt the cells
•We must start with a
tissue sample
containing the protein.
•Most proteins are
typically found within
a cell, so the tissue
must be subjected to a
homogenizing process
in order to break cell
walls and release
protein.

8. Remove debris
• Centrifugation of this extract
at moderately high speeds
sediments the nuclei,
mitochondria, membranes
and other insoluble material.
• The remainder of the
purification is an attempt to
separate the LDH contained in
the clear supernatant from
the other soluble proteins.
• The large centrifuge tube
must then be balanced before
it can be placed in the
instrument; this is done using
a separate tube filled with
water and a pan balance.

9. Ammonium Sulfate ppt
• One of the most commonly used crude purification
techniques involves the use of differential solubility.
• Proteins precipitate with increasing ammonium sulfate
concentrations, with most proteins precipitating
somewhere between 10% and 60% ammonium sulfate.
• This process depends on the physical or chemical
interaction between the protein and the precipitating
agent. In this lab, (NH4)2SO4 is used to precipitate out
LDH.

10. Dialysis
• Passage of solutes through a
semi-permeable membrane.
• Pores in the dialysis membrane
are of a certain size.
• Protein stays in; water, salts,
protein fragments, and other
molecules smaller than the pore
size pass through.
• We used dialysis in this lab to
remove the excess, unwanted
(NH4)2SO4 and other small
impurities while simultaneously
exchanging the extraction buffer
with dialysis buffer.

11. Affinity chromatography
• Most purification methods involve chromatography.
Chromatographic methods involve a column of an
insoluble material that can bind molecules based on
specific properties common to proteins.
• The solution containing the mixture of proteins is
then allowed to pass through the column; the
protein of interest may bind (depending on its
properties), while at least some impurities remain
in solution and leave the column. The procedure is
completed by eluting (i.e. “removing”) the proteins
that have bound to the column.
• In this lab we used a Cibacron blue affinity column
to purify LDH. This molecule mimics the shape and
charge characteristics of pyridine nucleotides to
which dehydrogenase proteins frequently bind to.

12. Analyzing the purity by SDS-
PAGE gel
• To determine which fractions from the
chromatography contain the protein.
SDS-PAGE gel electrophoresis is a good
method to use for determining the purity
of the protein.
• This method separates proteins according
their molecular weight and length of
polypeptide chain. Proteins all exhibit the
same charge per unit mass due to the
binding of SDS resulting in fractionation
by size and mass.

13. SDS PAGE for Purification

14. Result of the SDS-PAGE
Purified protein band

15. Future strategies
• Performing a Western Blot analysis to
confirm the presence of LDH in each
sample and fraction.
• Expression of Lactate Dehydrogenase
using recombinant DNA technology
and use of various “tags”.
• Execute Colorimetric Assays for
protein determination.
• Perform Gel filtration
chromatography.

16. Long term objectives
• Recognize the interactions with its
substrates by studying the enzyme
kinetics.
• Perform protein crystallization to unravel
the 3 D structure and relate its function
and structure.
• Analyze the effects of an inhibitor, and
studying the effects of chemical
modification of the enzyme.

17. Application of lactate
dehydrogenase
• LDH is often used as a marker of
tissue breakdown, therefore it can
be used in detection of myocardial
infarction, hemolysis.
• Variation in LDH isozymes profile
might have a role in studying
muscle response to training so its
used in sports medicine.

18. Thank you !