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Purification of Lactate DehydrogenasePurification of Lactate Dehydrogenase
Irene J. Wilson
Dominican University of California
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Wilson, Irene J., "Purification of Lactate Dehydrogenase" (2016). Scholarly and Creative Works Conference 2020. 67. https://scholar.dominican.edu/scw/scw2016/AllConference/
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Purification of Lactate Dehydrogenase
Irene J. Wilson
Dominican Scholarly and Creative Works Conference
Spring 2016
Introduction to Lactate Dehydrogenase (LDH)
● 3 genes provide instructions for building LDH (protein subunits of LDH):
○ lactate dehydrogenase A (LDHA) = M subunit
○ lactate dehydrogenase B (LDHB) = H subunit
○ lactate dehydrogenase C (LDHC)
Introduction to Lactate Dehydrogenase (LDH)
● Tetramer- composed of a combination of four subunits
○ Molecular weight of tetramer is about 140 kDa
● Tetramer made of 2 subunit types: M and H
○ Molecular weight of each subunit is about 35 kDa
● Five possible isoforms exist:
○ LDH 1 - (H 4 )
○ LDH 2 - (H 3 M)
○ LDH 3 - (H 2 M 2 )
○ LDH 4 - (HM 3 )
○ LDH 5 - (M 4 )
M subunit H subunit
Summary of Materials and Methods
● Goal of the project was to purify LDH from a cow heart
● Techniques used to purify LDH:
○ Homogenization
○ Centrifugation
○ Ammonium Sulfate Precipitation
○ Gel-filtration Chromatography
● Colorimetric Assay to quantify total protein yields
● Techniques used to verify presence of LDH:
○ LDH Enzyme Activity Assay
○ Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
○ Coomassie-stained Gel
○ Western Blot with anti-LDH antibody
Purification of LDH
● Homogenization, centrifugation, and ammonium sulfate precipitation
○ Creation of the pellet (B5), which is enriched in LDH
Colorimetric Assay to Quantify Total Protein Yields
● Measured absorbance of varying volumes of a BSA protein standard (1 μg/μL)
● Obtained best-fit line to determine the amount of protein in column fractions
Colorimetric Assay to Quantify Protein Yields
● Used BSA standard curve to calculate the total protein concentration of the
protein-containing fractions
● F1-F12 column fractions and B1-B3 and B5 centrifugation fractions contained
protein
Verification of LDH
● LDH Enzyme Activity Assay
● Measures the rate at which NAD
and lactate are converted to pyruvate and NADH
● Production of NADH monitored as increase in absorbance at 340 nm
Verification of LDH
● Assayed all protein-containing fractions
● F2-F6 column fractions and B1-B5 centrifugation fractions contained LDH
● Used the 2 column fractions with the highest rate of measured LDH activity (F
and F4) and the centrifugation fraction B5 (pellet) for visualization by SDS-PAGE
SDS-PAGE
(Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)
● SDS eliminates differences in protein shape and
native charge
○ All proteins migrate based on chain length/molecular
weight
● Gel was cut in half:
○ One half stained with Coomassie Blue stain to detect
presence of total proteins
○ One half transferred to a membrane via Western
blotting
Coomassie Blue Stain of SDS-PAGE Gel
Protein B5 LDH F3 F Marker Pellet Standard Sample Sample
Lanes: 1 2 3 4 5
LDH subunits at 35 kDa
Conclusion
● Goal of the project was to purify LDH from a cow heart
● LDH was successfully purified from a cow heart
○ Samples F3-F5 contained LDH
○ Purification can be improved
● Ideal for undergraduate academic setting:
○ Learn valuable laboratory techniques
○ Collect, analyze, and critique results
○ Practice responsibility and critical thinking
Acknowledgements
Dr. Ojeda and Dr. Spain
Dr. Hall
Faculty and Staff of the Department of Natural Sciences & Mathematics
Honors Program
My parents
