Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/9 July 2015"
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<u>Batch Purification</u> | <u>Batch Purification</u> | ||
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A new method of protein purification is discovered when searching online databases. Compared to gravity filtration through a nickel charged column, the advantages of this new method become apparent. Specifically, this method drastically decreases the amount of time needed to purify proteins, and allows for more homogenous mixing of sample with reagents added (binding, wash, and elution buffers). Batch purification shares the same general idea as column chromatography. Instead of a column, Histidine resin is placed in a Falcon tube and centrifugation is used to mix and separate contaminant proteins from our Tamura proteins. Instead of collecting what drips through the column as our fractions, after centrifugation, the supernatant is decanted from the tube and collected as fractions. | A new method of protein purification is discovered when searching online databases. Compared to gravity filtration through a nickel charged column, the advantages of this new method become apparent. Specifically, this method drastically decreases the amount of time needed to purify proteins, and allows for more homogenous mixing of sample with reagents added (binding, wash, and elution buffers). Batch purification shares the same general idea as column chromatography. Instead of a column, Histidine resin is placed in a Falcon tube and centrifugation is used to mix and separate contaminant proteins from our Tamura proteins. Instead of collecting what drips through the column as our fractions, after centrifugation, the supernatant is decanted from the tube and collected as fractions. | ||
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1. Chill a 50mL Falcon tube on ice. Add 2mL of resin slurry. | 1. Chill a 50mL Falcon tube on ice. Add 2mL of resin slurry. | ||
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2. Centrifuge the tube for 2 minutes at 700g. Remove supernatant. NOTE: The speed was increased to 1,000g and then 3,000g to maximize the amount of ethanol supernatant. | 2. Centrifuge the tube for 2 minutes at 700g. Remove supernatant. NOTE: The speed was increased to 1,000g and then 3,000g to maximize the amount of ethanol supernatant. | ||
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3. Add 2 bed volumes (BV) of 1X binding buffer to resin and mix thoroughly on a rotary spinner until buffer is fully suspended in resin. | 3. Add 2 bed volumes (BV) of 1X binding buffer to resin and mix thoroughly on a rotary spinner until buffer is fully suspended in resin. | ||
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4. Centrifuge tube for 2 minutes at 700g. Remove buffer. | 4. Centrifuge tube for 2 minutes at 700g. Remove buffer. | ||
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5. Add protein lysate diluted in 1X binding buffer in 1 to 1 volume ratio to tube. Mix for 30 minutes on an end over end rotation vehicle (rotary spinner). | 5. Add protein lysate diluted in 1X binding buffer in 1 to 1 volume ratio to tube. Mix for 30 minutes on an end over end rotation vehicle (rotary spinner). | ||
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6. Centrifuge the tube for 2 minutes at 700g. Decant supernatant and collect as Fraction 1. | 6. Centrifuge the tube for 2 minutes at 700g. Decant supernatant and collect as Fraction 1. | ||
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7. Wash with 2 BV's of 1X wash buffer (20 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 2. | 7. Wash with 2 BV's of 1X wash buffer (20 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 2. | ||
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8. Wash with 2 BV's of 1X wash buffer (50 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 3. | 8. Wash with 2 BV's of 1X wash buffer (50 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 3. | ||
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9. Elute using 1 BV of elution buffer (100 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction4. | 9. Elute using 1 BV of elution buffer (100 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction4. | ||
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10. Repeat elution with 250 mM imidazole solution. Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 5. | 10. Repeat elution with 250 mM imidazole solution. Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 5. | ||
Revision as of 18:05, 10 July 2015
A new method of protein purification is discovered when searching online databases. Compared to gravity filtration through a nickel charged column, the advantages of this new method become apparent. Specifically, this method drastically decreases the amount of time needed to purify proteins, and allows for more homogenous mixing of sample with reagents added (binding, wash, and elution buffers). Batch purification shares the same general idea as column chromatography. Instead of a column, Histidine resin is placed in a Falcon tube and centrifugation is used to mix and separate contaminant proteins from our Tamura proteins. Instead of collecting what drips through the column as our fractions, after centrifugation, the supernatant is decanted from the tube and collected as fractions.
Protocol
1. Chill a 50mL Falcon tube on ice. Add 2mL of resin slurry.
2. Centrifuge the tube for 2 minutes at 700g. Remove supernatant. NOTE: The speed was increased to 1,000g and then 3,000g to maximize the amount of ethanol supernatant.
3. Add 2 bed volumes (BV) of 1X binding buffer to resin and mix thoroughly on a rotary spinner until buffer is fully suspended in resin.
4. Centrifuge tube for 2 minutes at 700g. Remove buffer.
5. Add protein lysate diluted in 1X binding buffer in 1 to 1 volume ratio to tube. Mix for 30 minutes on an end over end rotation vehicle (rotary spinner).
6. Centrifuge the tube for 2 minutes at 700g. Decant supernatant and collect as Fraction 1.
7. Wash with 2 BV's of 1X wash buffer (20 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 2.
8. Wash with 2 BV's of 1X wash buffer (50 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 3.
9. Elute using 1 BV of elution buffer (100 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction4.
10. Repeat elution with 250 mM imidazole solution. Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 5.