NanoBio: Protein Purification

Materials

 * B-PER Bacterial Protein Extraction Reagent (Fisher catalog # PI78248, Pierce catalog # 78248)
 * HisTrap FF 1 mL (Fisher catalog # 17525501)

Bacterial Lysis

 * 1) Follow the instructions using the B-PER reagent.
 * 2) Add 2M imidazole to a final concentration of 20 mM.
 * 3) Filter the extracted protein solution through a 0.22 um syringe filter.

Buffer Preparation

 * All buffers should be passed through a 0.22 um filter and allowed to equilibrate at 4C overnight before use. These steps remove particles and air bubbles which can damage the column.

Preparation of the Lines

 * 1) Attach the appropriate (typically 2 mL) sample loop.
 * 2) Rinse the sample loop with water.
 * 3) Select Manual: Flowpath: Injection Valve: Load.
 * 4) Inject 2.5 mL Millipore water into the sample injection port. Make sure that you see drops entering the waste container.
 * 5) Repeat 2.5 mL Millipore water injection.
 * 6) Replace existing buffers in lines A2 and B with Millipore water.
 * 7) Place lines A2 and B in ~500 mL container of Millipore water.
 * 8) Select Manual:
 * 9) *Pump: Flow: 5mL/min
 * 10) *Pump: Gradient: 50% B, length of gradient 0
 * 11) *Flowpath: Injection Valve: Inject
 * 12) *Flowpath: Column Position: Bypass Column Position 1
 * 13) *Flowpath: Buffer Valve: A2
 * 14) *Other: Pause TimeOut: 50 mL accumulated volume
 * 15) Replace the water in lines A2 and B with low imidazole and high imidazole buffer, respectively.
 * 16) Place lines A2 and B in low imidazole and high imidazole buffer, respectively.
 * 17) Select Manual:
 * 18) *Pump: Flow: 5mL/min
 * 19) *Pump: Gradient: 0% B, length of gradient 0
 * 20) *Flowpath: Injection Valve: Inject
 * 21) *Flowpath: Column Position: Bypass Column Position 1
 * 22) *Flowpath: Buffer Valve: A2
 * 23) *Other: Pause TimeOut: 35 mL accumulated volume
 * 24) Select Manual:
 * 25) *Pump: Flow: 5mL/min
 * 26) *Pump: Gradient: 100% B, length of gradient 0
 * 27) *Flowpath: Injection Valve: Inject
 * 28) *Flowpath: Column Position: Bypass Column Position 1
 * 29) *Flowpath: Buffer Valve: A2
 * 30) *Other: Pause TimeOut: 35 mL accumulated volume

Column Washing & Equilibration

 * Note: the maximum backpressure that a HisTrap FF column can tolerate is 0.3 MPa
 * 1) Equilibrate the sample loop with the appropriate buffer.
 * 2) Load 2.5 mL of B-PER reagent (or buffer that your protein lysate is in) in a syringe. Inject into the sample port.
 * 3) Wash residue off a previously used column. Select Manual:
 * 4) *Pump: Flow: 1mL/min
 * 5) *Pump: Gradient: 100% B, length of gradient 0
 * 6) *Flowpath: Injection Valve: Inject
 * 7) *Flowpath: Column Position: Column 4 (or position of HisTrap column)
 * 8) *Flowpath: Buffer Valve: A2
 * 9) *Other: Pause TimeOut: 15 mL accumulated volume (for a 5 mL column; should be ~3 column volumes)
 * 10) Equilibrate the column. Select Manual:
 * 11) *Pump: Flow: 1mL/min
 * 12) *Pump: Gradient: 0% B, length of gradient 0
 * 13) *Flowpath: Injection Valve: Inject
 * 14) *Flowpath: Column Position: Column 4 (or position of HisTrap column)
 * 15) *Flowpath: Buffer Valve: A2
 * 16) *Other: Pause TimeOut: 50 mL accumulated volume (for a 5 mL column; should be ~10 column volumes)

Run Programmed Separation Program

 * 1) Check that the fraction collector is filled with tubes and correctly aligned.
 * 2) Load your sample into the sample loop.
 * 3) *Select Manual: Flowpath: Injection Valve: Load.
 * 4) *Inject your sample into the sample injection port.
 * 5) Run CAjoHisMethod for 5 mL columns and run CAjoHisMethod1mL for 1 mL columns.