Macromolecules Lab Report
Background: Lipid and protein composition varies in tissue types.
Results: When comparing liver, muscle, and adipose tissue, adipose tissue had the highest percent lipid and liver tissue had the highest percent protein.
Conclusion: Based on analysis of the gels, the liver appeared to have the most variety in proteins when comparing liver, muscle, and adipose tissue.
Significance: In order to determine the composition of a tissue, specific macromolecules can be extracted, quantified, and analyzed.
ABSTRACT Tissue types have a variety of compositions, and one way to describe these differences is by comparing macromolecule content. The four macromolecules – lipids, proteins, carbohydrates, and nucleic acids – can be quantified and analyzed. …show more content…
The Folch Method was used to extract lipids, and the Lowry Method was used to extract proteins. Gel electrophoresis and staining was used to analyze differences in protein composition in the three tissues. When comparing liver, skeletal muscle, and adipose tissue samples, the highest lipid content was in adipose tissue while the highest protein content was in the liver. The liver appeared to have the most variety in proteins while adipose tissue appeared to have the least variety in proteins.
INTRODUCTION Cells in the body are made up of four major macromolecules - lipids, proteins, carbohydrates, and nucleic acids (3).
Lipids are macromolecules made up of fatty acids connected by ester bonds. Proteins, the most abundant and versatile macromolecule, is made up of a chain of amino acids connected by peptide bonds. Carbohydrates are sugars made up of monosaccharides connected by glycosidic bonds. Nucleic acids are made up of nucleotides, and examples include DNA and RNA. Each tissue in the body has a different amounts of the four macromolecules. Based on a study done by Baker, the percentage of lipid in adipose tissue in humans at birth is about 40% and increases up to about 75% with age (1). According to Pawan and Clode, The percentage of protein in adipose tissue in humans is about 2.1% (6).
The Folch Method can be used to extract lipids from tissue samples. Lipids are hydrophobic and thus do not dissolve in polar substances such as water but can dissolve in organic solvents. The Lowry Method is a commonly used and highly reproducible method of protein …show more content…
determination. Gel electrophoresis is a method used to separate macromolecules based on size and charge. Smaller proteins travel farther down the gel while the larger proteins are unable to move through the crosslinks of the gel as quickly as the smaller proteins. Each gel consists of two layers, a stacking layer and a resolving layer. The resolving layer is the bottom layer of the gel that separates the protein based on size. The stacking layer is above the resolving layer and contains a lower acrylamide percentage. The stacking layer is where the protein is loaded and packed into one band.
The most common method of separation of proteins is polyacrylamide gel electrophoresis (PAGE). There are two types of PAGE: SDS-PAGE and Simple PAGE. Both types of PAGE gels consist of 30% acrylamide, Tris-HCl pH 8.0, 10% ammonium persulfate (APS), and TEMED. APS and TEMED initiate the polymerization of acrylamide to form a crosslinked gel. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) uses a gel that contains sodium dodecyl sulfate (SDS) in both the gel and sample loading buffer. The purpose of SDS is to denature the proteins to make the separation by polypeptide chain size possible and add a negative charge to the proteins. Simple polyacrylamide gel electrophoresis (Simple-PAGE) contains no detergent so proteins are not denatured and keep their form and activity.
Coomassie, a bright blue dye that stains proteins on gels non-specifically. It cannot stain for a specific protein but rather stains for all proteins present on a gel. The coomassie staining was used to detect differences between the proteins present in liver, muscle, and adipose tissue. Sigma Red staining was used to detect the activity of alkaline phosphatase. After staining to visualize the bands, the relative mobility of a band can be compared to the protein ladder to provide an estimate of the molecular mass of that protein. EXPERIMENTAL PROCEDURES
Lipid Extraction
Lipid extraction was carried out by using the Folch Method. Samples of liver, muscle, and adipose tissue weighing 0.10 g were obtained and a 1:1 mixture of methanol and chloroform was used to extract and homogenize the tissue samples. The homogenized tissue samples were placed on a vortex for 30 minutes. The samples were then filtered using filtered paper and 1mL of 0.9% NaCl was added to each of the filtrates. The samples were centrifuged for 10 minutes to separate the samples into two layers. The aqueous top layer was disposed and the remaining layer was dried under a stream of nitrogen to evaporate any remaining chloroform. The dried samples were weighed to determine the percent lipid in each of the liver, muscle, and adipose tissue samples.
Protein Extraction Samples of muscle, liver, and adipose tissue weighing 0.51 g were obtained and 9 volumes of 1X PBS with 1mM EDTA were added to the samples to be homogenized. The homogenized samples were centrifuged for 20 minutes at 4,000 rpm. A portion of each sample was diluted with buffer. The liver tissue was diluted 1:100, the muscle tissue was diluted 1:50, the adipose tissue was diluted 1:10, and the alkaline phosphatase was diluted 1:4 with a final total volume of 1.5 mL.
Protein Quantification The Lowry Method was used to quantify proteins in the samples. A standard curve protein assay was prepared using 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, and 0.5 mg/mL Bradford Protein Assay (BSA) to make 0.5 mL of each standard. Lowry Reagent I was prepared adding 49 mL of 4% sodium carbonate, 1 mL of 1% copper sulfate, and 1 mL of 2% sodium potassium tartrate to 49 mL of 0.2 M NaOH. 2.5 mL of Lowry Reagent I was added to each standard and incubated at room temperature for 10 minutes. Lowry reagent II was prepared by diluting the Folin-Ciocalteu 1:1 with water. 0.25 mL of Lowry Reagent II was added to each standard, mixed immediately to prevent degradation of Lowry Reagent II, and incubated for 30 minutes at room temperature. The absorbance at each standard concentration was measured at 750 nm. The protein concentrations of the samples could then be measured by comparing the sample absorbances to the standard curve.
Gel Electrophoresis
Gel electrophoresis is a method used to separate macromolecules based on size and charge.
Polyacrylamide gel electrophoresis (PAGE) is a commonly used method for separating proteins. Both SDS-PAGE and Simple-PAGE gels were prepared. The resolving layers for the SDS-PAGE and Simple-PAGE gels were prepared with 3.25mL water, 2.0mL 4X resolving buffer, 2.7mL 40% acrylamide solution, 80μL 10% APS, and 3μL TEMED. The stacking layers for the SDS-PAGE and Simple-PAGE gels were prepared with 4.6mL water, 2.0mL 4X stacking buffer, 1.3mL 40% acrylamide solution, 48μL 10% APS, and 5μL TEMED. The SDS-PAGE gel used the buffers containing SDS and the Simple-PAGE gels used the buffers without SDS. The gels were run at 100 V for about 60
minutes.
Detection of Proteins in PAGE Gels
To stain the gel with coomassie, enough coomassie stain was poured onto the gels to completely submerge them and the gels were incubated at room temperature for 10 minutes on a rocker. A coomassie destain solution was poured onto the gels until the individual bands were visible and the gels were clear.
To stain the gels with Sigma Red, the staining solution was prepared adding one Tris buffer tablet and one sigma red tablet to 10 mL of water. The staining solution was poured over the gels to and incubated at room temperature on a rocker until a red band was visible. The gels were photographed under UV light to detect the protein bands.
Results: When comparing liver, muscle, and adipose tissue, adipose tissue had the highest percent lipid and liver tissue had the highest percent protein.
Conclusion: Based on analysis of the gels, the liver appeared to have the most variety in proteins when comparing liver, muscle, and adipose tissue.
Significance: In order to determine the composition of a tissue, specific macromolecules can be extracted, quantified, and analyzed.
ABSTRACT Tissue types have a variety of compositions, and one way to describe these differences is by comparing macromolecule content. The four macromolecules – lipids, proteins, carbohydrates, and nucleic acids – can be quantified and analyzed. …show more content…
The Folch Method was used to extract lipids, and the Lowry Method was used to extract proteins. Gel electrophoresis and staining was used to analyze differences in protein composition in the three tissues. When comparing liver, skeletal muscle, and adipose tissue samples, the highest lipid content was in adipose tissue while the highest protein content was in the liver. The liver appeared to have the most variety in proteins while adipose tissue appeared to have the least variety in proteins.
INTRODUCTION Cells in the body are made up of four major macromolecules - lipids, proteins, carbohydrates, and nucleic acids (3).
Lipids are macromolecules made up of fatty acids connected by ester bonds. Proteins, the most abundant and versatile macromolecule, is made up of a chain of amino acids connected by peptide bonds. Carbohydrates are sugars made up of monosaccharides connected by glycosidic bonds. Nucleic acids are made up of nucleotides, and examples include DNA and RNA. Each tissue in the body has a different amounts of the four macromolecules. Based on a study done by Baker, the percentage of lipid in adipose tissue in humans at birth is about 40% and increases up to about 75% with age (1). According to Pawan and Clode, The percentage of protein in adipose tissue in humans is about 2.1% (6).
The Folch Method can be used to extract lipids from tissue samples. Lipids are hydrophobic and thus do not dissolve in polar substances such as water but can dissolve in organic solvents. The Lowry Method is a commonly used and highly reproducible method of protein …show more content…
determination. Gel electrophoresis is a method used to separate macromolecules based on size and charge. Smaller proteins travel farther down the gel while the larger proteins are unable to move through the crosslinks of the gel as quickly as the smaller proteins. Each gel consists of two layers, a stacking layer and a resolving layer. The resolving layer is the bottom layer of the gel that separates the protein based on size. The stacking layer is above the resolving layer and contains a lower acrylamide percentage. The stacking layer is where the protein is loaded and packed into one band.
The most common method of separation of proteins is polyacrylamide gel electrophoresis (PAGE). There are two types of PAGE: SDS-PAGE and Simple PAGE. Both types of PAGE gels consist of 30% acrylamide, Tris-HCl pH 8.0, 10% ammonium persulfate (APS), and TEMED. APS and TEMED initiate the polymerization of acrylamide to form a crosslinked gel. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) uses a gel that contains sodium dodecyl sulfate (SDS) in both the gel and sample loading buffer. The purpose of SDS is to denature the proteins to make the separation by polypeptide chain size possible and add a negative charge to the proteins. Simple polyacrylamide gel electrophoresis (Simple-PAGE) contains no detergent so proteins are not denatured and keep their form and activity.
Coomassie, a bright blue dye that stains proteins on gels non-specifically. It cannot stain for a specific protein but rather stains for all proteins present on a gel. The coomassie staining was used to detect differences between the proteins present in liver, muscle, and adipose tissue. Sigma Red staining was used to detect the activity of alkaline phosphatase. After staining to visualize the bands, the relative mobility of a band can be compared to the protein ladder to provide an estimate of the molecular mass of that protein. EXPERIMENTAL PROCEDURES
Lipid Extraction
Lipid extraction was carried out by using the Folch Method. Samples of liver, muscle, and adipose tissue weighing 0.10 g were obtained and a 1:1 mixture of methanol and chloroform was used to extract and homogenize the tissue samples. The homogenized tissue samples were placed on a vortex for 30 minutes. The samples were then filtered using filtered paper and 1mL of 0.9% NaCl was added to each of the filtrates. The samples were centrifuged for 10 minutes to separate the samples into two layers. The aqueous top layer was disposed and the remaining layer was dried under a stream of nitrogen to evaporate any remaining chloroform. The dried samples were weighed to determine the percent lipid in each of the liver, muscle, and adipose tissue samples.
Protein Extraction Samples of muscle, liver, and adipose tissue weighing 0.51 g were obtained and 9 volumes of 1X PBS with 1mM EDTA were added to the samples to be homogenized. The homogenized samples were centrifuged for 20 minutes at 4,000 rpm. A portion of each sample was diluted with buffer. The liver tissue was diluted 1:100, the muscle tissue was diluted 1:50, the adipose tissue was diluted 1:10, and the alkaline phosphatase was diluted 1:4 with a final total volume of 1.5 mL.
Protein Quantification The Lowry Method was used to quantify proteins in the samples. A standard curve protein assay was prepared using 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, and 0.5 mg/mL Bradford Protein Assay (BSA) to make 0.5 mL of each standard. Lowry Reagent I was prepared adding 49 mL of 4% sodium carbonate, 1 mL of 1% copper sulfate, and 1 mL of 2% sodium potassium tartrate to 49 mL of 0.2 M NaOH. 2.5 mL of Lowry Reagent I was added to each standard and incubated at room temperature for 10 minutes. Lowry reagent II was prepared by diluting the Folin-Ciocalteu 1:1 with water. 0.25 mL of Lowry Reagent II was added to each standard, mixed immediately to prevent degradation of Lowry Reagent II, and incubated for 30 minutes at room temperature. The absorbance at each standard concentration was measured at 750 nm. The protein concentrations of the samples could then be measured by comparing the sample absorbances to the standard curve.
Gel Electrophoresis
Gel electrophoresis is a method used to separate macromolecules based on size and charge.
Polyacrylamide gel electrophoresis (PAGE) is a commonly used method for separating proteins. Both SDS-PAGE and Simple-PAGE gels were prepared. The resolving layers for the SDS-PAGE and Simple-PAGE gels were prepared with 3.25mL water, 2.0mL 4X resolving buffer, 2.7mL 40% acrylamide solution, 80μL 10% APS, and 3μL TEMED. The stacking layers for the SDS-PAGE and Simple-PAGE gels were prepared with 4.6mL water, 2.0mL 4X stacking buffer, 1.3mL 40% acrylamide solution, 48μL 10% APS, and 5μL TEMED. The SDS-PAGE gel used the buffers containing SDS and the Simple-PAGE gels used the buffers without SDS. The gels were run at 100 V for about 60
minutes.
Detection of Proteins in PAGE Gels
To stain the gel with coomassie, enough coomassie stain was poured onto the gels to completely submerge them and the gels were incubated at room temperature for 10 minutes on a rocker. A coomassie destain solution was poured onto the gels until the individual bands were visible and the gels were clear.
To stain the gels with Sigma Red, the staining solution was prepared adding one Tris buffer tablet and one sigma red tablet to 10 mL of water. The staining solution was poured over the gels to and incubated at room temperature on a rocker until a red band was visible. The gels were photographed under UV light to detect the protein bands.