Upload
programabric
View
851
Download
0
Embed Size (px)
DESCRIPTION
Citation preview
Extraction and Quantification of Proteins in Syzygium jambos used as
Diabetic Adjuvants in Puerto Rico
Yolanda I. Rodríguez-Rivera BIOL 4990
Mentor: Dr. Jannette Gavillán-Suárez
Proteins
• Proteins are biochemical compounds consisting of polypeptides that facilitate a biological function.
Protein Functions
Ramírez Ramírez, G, Heil, M, Orona Tamayo, D, Adame Álvarez
RM, Ramírez Ramírez, DT, Durán Flores, FD. 2008. ANÁLISIS DE
PROTEÍNAS EN GRÁNULOS Y HOJAS DE Acacia hindsii Y EN
HORMIGAS DE LA REGIÓN DE IRAPUATO . Guanajuato (MX):
Universidad Autónoma de Querétaro (MX). Disponible en:
http://www.uaq.mx/investigacion/difusion/veranos/memorias-
2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf
Solubility of Proteins
Hydrophilic and Hydrophobic residues on surface
Charged and polar surfaces will have higher solubility
Previous studies A study shows that a protein named ADMc1 in
the seeds of Momordica charantia (cundiamor) has hypoglycemic/anti-hyperglycemic properties.
United States Patent Application Publication, Antidiabetic Protein: Pub.No: US
2011/0046054
Previous studies
• ADMc1 was found highly effective and needed to be administered only once a day to maintain normal blood glucose levels.
• The protein was detected and purified from the seeds of the fruit the plant provides.
• Published work states that the polypeptide-p is made of 166 amino acid residues and said to be of 11 kDa.
Specific aims of this study
To extract proteins on leaves of Syszygium jambos (Pomarrosa del río).
To quantify the proteins by the Lowry method.
Relevance
Determination of proteins that could serve as biomarkers of hypoglycemic/anti-hyperglycemic activities for the reduction of blood glucose in diabetic patients.
Methodology : Extraction of Proteins
3 replicates of the plant
Wash pellet with acetone, 10% TCA and 80% acetone.
Centrifuge.
2 g of of dry pellet is
resuspended in Phenol
Add dense SDS buffer and centrifuge
Separate phases and precipitate Ph phase with methanol and
ammonium acetate.
Recover insoluble
proteins by centrifugation
Wash 3 times with methanolic
ammonium acetate.
Wash with 80% acetone
Quantify proteins in pellet and
supernatant
Methodology: Protein Quantification
BSA Concentration and Calibration
Curve
Add BSA [0, 20, 40, 60, 80, 100 µl] to
these tubes & 100 µl of unknowns
Add 2 ml of Lowry Solution to each one of the test
tubes
Incubate at room temperature for 30
minutes
Add 0.2 ml of diluted Folin-
phenol solution to each tube
Incubate 10 minutes at room
temperature
Vortex each tube immediately
Determine absorbance of each sample at 600 nm
Plot absorbance vs mg BSA to obtain a
calibration curve
Lowry colorimetric reaction
Results
y = 7.7027x + 0.0333 R² = 0.9939
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04
Ab
sorb
ance
(6
00
nm
)
BSA Concentration (mg/ml)
BSA Calibration Curve
Results for Syszygium jambos supernatant
• Concentrations of unknowns:
– X1 = 0.33 mg/ml
– X2 = 0.57 mg/ml
– X3 = 0.44 mg/ml
[Protein] = 0.45 mg/ml of supernatant
[Protein] = 0.23 mg/g dry weight
In comparison with:
[Protein] = 0.25 mg/g dry weight (Gómez-Vidal, et.al)
Analysis of Results
• The amount of protein found in this assay is comparable to results in Gómez-Vidal, S, et.al.
• Given that they were found in the supernatant, they could be protein inside the cell.
• The proteins found on the pellet, which are insoluble, are probably structural and could pertain to the cell wall.
Future Work
• Analyze protein in leaves of studied plants and categorize them by their function via 2-dimensional electrophoresis.
• Extract and quantify proteins in leaves of Tapeinochious annasae, Costus speciousus and Tradescantia spathacea.
References
Gómez-Vidal S, Tena M, López-Llorca LV, Salinas J (2007). Protein Extraction from
Phoenix dactylifera L. leaves, a recalcitrant material, for two dimensional electrophoresis. Electrophoresis, 29: 448-456.
Hurkman WJ, Tanaka CK (1986). Solubilization of Plant Membrane Proteins for Analysis by Two-Dimensional Gel Electrophoresis. Plant Physiol., 81: 802-806.
Biology at Davidson [Internet]. 2000. Biology at Davidson in North Carolina. Protein Determination - Lowry Procedure; 2011 Sep 16. Available from: http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html
Ramírez Ramírez, G, Heil, M, Orona Tamayo, D, Adame Álvarez RM, Ramírez Ramírez, DT, Durán Flores, FD [Internet]. 2008. ANÁLISIS DE PROTEÍNAS EN GRÁNULOS Y HOJAS DE Acacia hindsii Y EN HORMIGAS DE LA REGIÓN DE IRAPUATO . Guanajuato (MX): Universidad Autónoma de Querétaro (MX). Disponible en: http://www.uaq.mx/investigacion/difusion/veranos/memorias-2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf
Extraction and Quantification of Proteins in Syzygium jambos used as
Diabetic Adjuvants in Puerto Rico
Yolanda I. Rodríguez-Rivera BIOL 4990
Mentor: Dr. Jannette Gavillán-Suárez