Kafatos:His-tagged Protein Purification (denaturing): Difference between revisions
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{{Kafatos/Christophides Lab}} | {{Kafatos/Christophides Lab-Small}} | ||
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# Pick a single colony of bacteria transformed with a His-tagged protein of interest into 1.5 mL of culture media containing both | # Pick a single colony of bacteria transformed with a His-tagged protein of interest into 1.5 mL of culture media containing both [[Ampicillin]] (100 μg/mL) and [[Chloramphenicol]] (34 μg/mL). Grow the cultures overnight. <BR> | ||
# Inoculate 10 mL of room temperature medium (including antibiotics) with 500 μL of the overnight cultures, and grow at 37°C for 60 minutes, with vigorous shaking.<BR> | # Inoculate 10 mL of room temperature medium (including antibiotics) with 500 μL of the overnight cultures, and grow at 37°C for 60 minutes, with vigorous shaking.<BR> | ||
# Take 500 µL of culture as an un-induced sample. Induce expression by adding IPTG to a final concentration of 1 mM (10 µL of 1 M stock made in water). Resuspend this pellet in 25 µL [[kafatos:4x SDS PAGE Reducing Sample Buffer|1x SDS PAGE reducing sample buffer]] and freeze.<BR> | # Take 500 µL of culture as an un-induced sample. Induce expression by adding IPTG to a final concentration of 1 mM (10 µL of 1 M stock made in water). Resuspend this pellet in 25 µL [[kafatos:4x SDS PAGE Reducing Sample Buffer|1x SDS PAGE reducing sample buffer]] and freeze.<BR> | ||
# Grow the cultures for an additional 5 hours, and make another sample from 500 µL cells as in step 3. Then transfer the remainder of the culture to microcentrifuge tubes putting 1 mL into each tube. Harvest the cells by centrifugation for 1 minute at 15,000x g, and discard supernatants.<BR> | # Grow the cultures for an additional 5 hours, and make another sample from 500 µL cells as in step 3. Then transfer the remainder of the culture to microcentrifuge tubes putting 1 mL into each tube. Harvest the cells by centrifugation for 1 minute at 15,000x g, and discard supernatants.<BR> | ||
# Resuspend 4 tubes of cells in 400 μL buffer B total (10x). Resuspend one tube, and then transfer to the next until all 4 are combined in the 400 µL volume. Lyse cells by pipetting them up and down gently vortexing, taking care to avoid frothing. The solution should become translucent when lysis is complete.<BR> | # Resuspend 4 tubes of cells in 400 μL buffer B total (10x). Resuspend one tube, and then transfer to the next until all 4 are combined in the 400 µL volume. Lyse cells by pipetting them up and down gently vortexing, taking care to avoid frothing. The solution should become translucent when lysis is complete. Freeze the remaining tubes for [[kafatos:His purification native conditions|purification under native conditions]].<BR> | ||
# Centrifuge the lysate for 20 minutes at 15,000x g to remove cellular debris, and transfer the supernatant to a fresh tube.<BR> | # Centrifuge the lysate for 20 minutes at 15,000x g to remove cellular debris, and transfer the supernatant to a fresh tube.<BR> | ||
# Put 500 µL of Ni-NTA slurry into a disposable column. Equilibrate with 1mL of buffer B. Let stand until lysates are done spinning, and then let the buffer drain through the column and discard.<BR> | # Put 500 µL of Ni-NTA slurry into a disposable column. Equilibrate with 1mL of buffer B. Let stand until lysates are done spinning, and then let the buffer drain through the column and discard.<BR> | ||
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# Wash the column with 2x 1 mL of buffer C. Save washes individually as w1 & w2.<BR> | # Wash the column with 2x 1 mL of buffer C. Save washes individually as w1 & w2.<BR> | ||
# Elute the protein with 5x 200 µL of buffer E. Save eluate fractions individually as e1-5.<BR> | # Elute the protein with 5x 200 µL of buffer E. Save eluate fractions individually as e1-5.<BR> | ||
# Add 25 µL of 4x SDS Page sample buffer to 75 µL of each sample. Heat all samples to 95ºC for 5 minutes and load 10 µL of samples on an SDS Page gel. | # Add 25 µL of 4x SDS Page sample buffer to 75 µL of each sample. Heat all samples to 95ºC for 5 minutes and load 10 µL of samples on an SDS Page gel. | ||
==Denaturing Buffer 300 mL== | |||
{| style="width:50%" | |||
| 100 mM NaH₂PO₄ || 4.2 g NaH₂PO₄ (FW 137.99) | |||
|- | |||
| 10 mM Tris-Cl || 0.36g Tris Base (FW 121.1) | |||
|- | |||
| 8 M Urea || 144 g (FW 60.06) | |||
|} | |||
Add water to 300 mL and set the pH appropriately for 100 mL portions.<BR> | |||
<B>Buffer B – 100 mL</B><BR> | |||
pH 8.0 using NaOH | |||
<B>Buffer C – 100 mL</B><BR> | |||
pH 6.3 using HCl (no adjustment was required) | |||
<B>Buffer E – 100 mL</B><BR> | |||
pH 4.5 using HCl | |||
Store at 4ºC and <font color="red">always adjust the pH prior to using</font><BR> | |||
Latest revision as of 04:00, 8 August 2006
His-tagged Protein Purification Mini Prep - Denaturing Conditions
- Pick a single colony of bacteria transformed with a His-tagged protein of interest into 1.5 mL of culture media containing both Ampicillin (100 μg/mL) and Chloramphenicol (34 μg/mL). Grow the cultures overnight.
- Inoculate 10 mL of room temperature medium (including antibiotics) with 500 μL of the overnight cultures, and grow at 37°C for 60 minutes, with vigorous shaking.
- Take 500 µL of culture as an un-induced sample. Induce expression by adding IPTG to a final concentration of 1 mM (10 µL of 1 M stock made in water). Resuspend this pellet in 25 µL 1x SDS PAGE reducing sample buffer and freeze.
- Grow the cultures for an additional 5 hours, and make another sample from 500 µL cells as in step 3. Then transfer the remainder of the culture to microcentrifuge tubes putting 1 mL into each tube. Harvest the cells by centrifugation for 1 minute at 15,000x g, and discard supernatants.
- Resuspend 4 tubes of cells in 400 μL buffer B total (10x). Resuspend one tube, and then transfer to the next until all 4 are combined in the 400 µL volume. Lyse cells by pipetting them up and down gently vortexing, taking care to avoid frothing. The solution should become translucent when lysis is complete. Freeze the remaining tubes for purification under native conditions.
- Centrifuge the lysate for 20 minutes at 15,000x g to remove cellular debris, and transfer the supernatant to a fresh tube.
- Put 500 µL of Ni-NTA slurry into a disposable column. Equilibrate with 1mL of buffer B. Let stand until lysates are done spinning, and then let the buffer drain through the column and discard.
- Load the cleared lysate supernatant containing the 6xHis-tagged protein onto the equilibrated Ni-NTA column (save the flow-through).
- Wash the column with 2x 1 mL of buffer C. Save washes individually as w1 & w2.
- Elute the protein with 5x 200 µL of buffer E. Save eluate fractions individually as e1-5.
- Add 25 µL of 4x SDS Page sample buffer to 75 µL of each sample. Heat all samples to 95ºC for 5 minutes and load 10 µL of samples on an SDS Page gel.
Denaturing Buffer 300 mL
| 100 mM NaH₂PO₄ | 4.2 g NaH₂PO₄ (FW 137.99) |
| 10 mM Tris-Cl | 0.36g Tris Base (FW 121.1) |
| 8 M Urea | 144 g (FW 60.06) |
Add water to 300 mL and set the pH appropriately for 100 mL portions.
Buffer B – 100 mL
pH 8.0 using NaOH
Buffer C – 100 mL
pH 6.3 using HCl (no adjustment was required)
Buffer E – 100 mL
pH 4.5 using HCl
Store at 4ºC and always adjust the pH prior to using
