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N-Acetyl-Hexosaminidase Lab Report

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The aims of this experiment were to separate and locate the enzyme N-acetyl-β-D-Hexosaminidase from protein sample #15 and determine the specific activity of the enzyme within the sample. DEAE cellulose anion exchange chromatography was used to separate proteins from within sample #15. The initial separation was performed through the addition of Tris/HCl buffer pH 7.2 and yielded a peak consisting of positively charged proteins. Salting out was performed with Tris/HCl pH 7.2 1.0 M salt buffer and eluted negatively charged proteins. Peaks also represented the highest amount of a negatively and positively charged protein respectively. It was determined that sample #15 did not contain N-acetyl-β-D-hexosaminidase. Sample #15 concentration was determined to be 4.7 mg/mL. INTRODUCTION Aside from the cellular role proteins have numerous uses ranging from cosmetics to clinical applications (1). To successfully add on to the current uses of proteins, successful separation and location of proteins must be performed. One method to achieve this is through column chromatography. Column chromatography is a biochemical technique, which can achieve …show more content…

In initial elution using 25 mM Tris/HCl pH 7.2 buffer test tube #10 was determined to have the highest peak with an absorbance of 0.12. In elution with 25 mM Tris/HCl pH 7.2 1.0 M salt buffer test tube #20 was determined to have the highest peak with an absorbance of 0.4. These peaks represent the amount of protein present within the fraction. The first peak may include proteins with many positive amino acid residues. The second peak contained proteins with negatively charged amino acid residues. Due to the differences in absorbance of test tubes #10 and #20, elution using the 25 mM Tris/HCl pH 7.2 1.0 M salt buffer eluted more proteins from the column than the 25 mM Tris/HCl pH 7.2 buffer. The concentration of the protein was determined to be 4.37

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A: Isolate HHHHHHH Method: Ni-Affinity Chromatography Reason: Six histadine amino acids at the end of the protein can bind to nickel very tightly. Nickel can bind to agarose bead very tightly. Use this strong affinity column we can isolate our protein, which has seven histadine amino acids. Buffer condition: His-binding buffer: • 50 mM Tris-HCl (pH8.0) 
 • 5 mM Imidazole 
 • 100 mM NaCl 
 • 0.1 mM EDTA 
 • 1 mM PMSF made fresh 
 His-wash buffer: • 50 mM Tris-HCl (pH8.0) 
 • 300 mM NaCl 
 • 15 mM Imidazole 
 • 0.1 mM EDTA 
 • 1 mM PMSF made fresh 
 is-elution buffer: • 50 mM Tris-Cl (pH8.0) 
 • 50 mM NaCl 
 • 300 mM Imidazole 
 • 0.1 mM EDTA 
 • 1 mM PMSF made fresh 
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