community.elabeln.com/experiments.php?mode=edit&id=314
#314
Protein Analysis
Complete
Purif: His₆-MBP-GFP / BL21(DE3) / Ni-NTA
This template captures Ni-NTA affinity purification of a recombinant His₆-MBP-GFP fusion protein (~72 kDa) from a 1 L IPTG-induced E. coli BL21(DE3) culture, supporting downstream crystallography and binding-assay work.
Goal: isolate >5 mg of >95% pure His₆-MBP-GFP fusion at >1 mg/mL concentration suitable for crystallography screens, biophysical characterization (DSF, ITC, SPR), and functional fluorescence-based assays.
Method overview: 1 L of LB+Kan culture inoculated to OD₆₀₀ 0.05 from overnight starter, grown at 37°C / 200 rpm to OD₆₀₀ 0.6-0.8, induced with 0.5 mM IPTG, shifted to 16°C and grown 18 hours for low-temperature folding. Cells harvested by centrifugation (6,000 × g × 15 min), resuspended in 50 mL lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 1× protease inhibitor cocktail, 0.1 mg/mL lysozyme), sonicated on ice (30 s on / 30 s off × 6 cycles), clarified at 30,000 × g × 30 min. Clarified lysate loaded onto pre-equilibrated 5 mL HisTrap HP at 1 mL/min on ÄKTA pure, washed with 20 mM and 50 mM imidazole steps, eluted with 250 mM imidazole, 1.5 mL fractions collected. Pooled elution fractions dialyzed against storage buffer (25 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT), concentrated to ~3 mg/mL via Amicon 30 kDa MWCO. QC by Bradford assay and 10% SDS-PAGE with Coomassie staining.
Outcome: 8.2 mg total yield from 1 L culture (4 mL final at 2.05 mg/mL), >95% purity by SDS-PAGE densitometry, single ~72 kDa band, identity verified by mass spectrometry intact-mass measurement (observed 71,842 Da; expected 71,840 Da). Suitable for crystallography setup.
Last saved: 34 seconds ago
12 fields loaded from His-tag Protein Purification baseline (SOP v2.3)
Expression
Host strain
select
BL21(DE3), Rosetta, Origami, HEK293, Sf9, CHO
Construct
text
pET28a-His₆-MBP-GFP
IPTG conc
number
0.5mM
Induction T / t
text
16 °C × 18 h
Lysis & Capture
Lysis method
select
sonication, French press, microfluidizer, detergent
Column
select
HisTrap HP 5 mL, Ni-NTA agarose, IMAC superflow
Flow rate
number
1.0mL/min
Imidazole steps
text
10 / 20 / 50 / 250 mM
Elution & QC
Eluate volume
number
4.0mL (post-dialysis)
Concentration
number
2.05mg/mL
Total yield
number
8.2mg
Purity (SDS-PAGE)
number
95%
01
3:30
Inoculate 1 L LB+Kan with 10 mL overnight starter at OD₆₀₀ 0.05, grow 37 °C / 200 rpm
02
18:00
Induce with 0.5 mM IPTG at OD₆₀₀ 0.7, shift to 16 °C, grow 18 h
03
0:25
Harvest by centrifugation 6,000 × g × 15 min, pellet stored -80 °C if not processed same day
04
0:20
Resuspend pellet in 50 mL lysis buffer + lysozyme + protease inhibitors
05
0:08
Sonicate on ice: 30 s on / 30 s off × 6 cycles, 40% amplitude
06
0:45
Clarify at 30,000 × g × 30 min, filter 0.45 µm
07
1:30
Equilibrate 5 mL HisTrap HP, load lysate at 1 mL/min, monitor A₂₈₀
08
1:20
Wash steps: 10 CV 20 mM imid, 5 CV 50 mM imid; elute with 5 CV 250 mM imid, collect 1.5 mL fracs
09
2:30
SDS-PAGE QC: lysate / FT / wash / elution fractions; Bradford assay on pooled fractions
10
5:00
Dialyze pooled elution → storage buffer 4 h, concentrate to ~3 mg/mL via Amicon 30 K, archive at -80 °C
| A | B | C | D | E | F | |
|---|---|---|---|---|---|---|
| 1 | Fraction | Vol (mL) | A₂₈₀ | Conc (mg/mL) | SDS-PAGE | Decision |
| 2 | FT | 60 | 0.42 | 0.18 | many bands | discard |
| 3 | W1 20 mM | 40 | 0.21 | 0.09 | few contaminants | discard |
| 4 | W2 50 mM | 25 | 0.08 | 0.03 | very faint | discard |
| 5 | E1 | 1.5 | 0.32 | 0.14 | target faint | discard (front) |
| 6 | E2 | 1.5 | 0.88 | 0.39 | target + 1 contam | pool conditionally |
| 7 | E3 | 1.5 | 2.41 | 1.07 | clean 72 kDa | pool |
| 8 | E4 | 1.5 | 3.05 | 1.36 | clean 72 kDa | pool |
| 9 | E5 | 1.5 | 3.41 | 1.52 | clean 72 kDa | pool |
| 10 | E6 | 1.5 | 2.18 | 0.97 | clean 72 kDa | pool |
| 11 | E7 | 1.5 | 1.04 | 0.46 | clean 72 kDa | pool |
| 12 | E8 | 1.5 | 0.51 | 0.23 | clean 72 kDa | pool |
EXP-2026-0524
BL21(DE3) transformation + clone selection
Y. Nakamura
May 24
EXP-2026-0610
His₆-MBP-GFP crystallography screen (1024)
Y. Nakamura
Jun 10
SOP-0623
His-tag Protein Purification SOP v2.3
QA Team
Jun 2
PLM-0124
pET28a-His₆-MBP-GFP construct sequence + map
Plasmid DB
Jan 15
AKT-MET
ÄKTA HisTrap method file (UNICORN)
Instrument Docs
Oct 2
Visibility
Nakamura LabY. Nakamura
Can write
Y. NakamuraDr. L. Wright
Use Ctrl + click to add text
{
"experiment": {
"id": 314,
"custom_id": "PURIF-MBP-GFP-2026-044",
"title": "Purif: His₆-MBP-GFP / BL21(DE3) / Ni-NTA",
"template": "Protein Purification",
"template_version": "v3.2",
"category": "Protein Analysis",
"status": "Complete",
"started_on": "2026-07-15",
"tags": [
"protein-purification",
"His-tag",
"Ni-NTA",
"BL21",
"recombinant-protein"
]
},
"main_text": "This template captures Ni-NTA affinity purification of a recombinant His₆-MBP-GFP fusion protein (~72 kDa) from a 1 L IPTG-induced E. coli BL21(DE3) culture, supporting downstream crystallography and binding-assay work.\nGoal: isolate >5 mg of >95% pure His₆-MBP-GFP fusion at >1 mg/mL concentration suitable for crystallography screens, biophysical characterization (DSF, ITC, SPR), and functional fluorescence-based assays.\nMethod overview: 1 L of LB+Kan culture inoculated to OD₆₀₀ 0.05 from overnight starter, grown at 37°C / 200 rpm to OD₆₀₀ 0.6-0.8, induced with 0.5 mM IPTG, shifted to 16°C and grown 18 hours for low-temperature folding. Cells harvested by centrifugation (6,000 × g × 15 min), resuspended in 50 mL lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 1× protease inhibitor cocktail, 0.1 mg/mL lysozyme), sonicated on ice (30 s on / 30 s off × 6 cycles), clarified at 30,000 × g × 30 min. Clarified lysate loaded onto pre-equilibrated 5 mL HisTrap HP at 1 mL/min on ÄKTA pure, washed with 20 mM and 50 mM imidazole steps, eluted with 250 mM imidazole, 1.5 mL fractions collected. Pooled elution fractions dialyzed against storage buffer (25 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT), concentrated to ~3 mg/mL via Amicon 30 kDa MWCO. QC by Bradford assay and 10% SDS-PAGE with Coomassie staining.\nOutcome: 8.2 mg total yield from 1 L culture (4 mL final at 2.05 mg/mL), >95% purity by SDS-PAGE densitometry, single ~72 kDa band, identity verified by mass spectrometry intact-mass measurement (observed 71,842 Da; expected 71,840 Da). Suitable for crystallography setup.",
"extra_fields": {
"Expression": {
"Host strain": {
"type": "select",
"value": "BL21(DE3), Rosetta, Origami, HEK293, Sf9, CHO"
},
"Construct": {
"type": "text",
"value": "pET28a-His₆-MBP-GFP"
},
"IPTG conc": {
"type": "number",
"value": "0.5 mM"
},
"Induction T / t": {
"type": "text",
"value": "16 °C × 18 h"
}
},
"Lysis & Capture": {
"Lysis method": {
"type": "select",
"value": "sonication, French press, microfluidizer, detergent"
},
"Column": {
"type": "select",
"value": "HisTrap HP 5 mL, Ni-NTA agarose, IMAC superflow"
},
"Flow rate": {
"type": "number",
"value": "1.0 mL/min"
},
"Imidazole steps": {
"type": "text",
"value": "10 / 20 / 50 / 250 mM"
}
},
"Elution & QC": {
"Eluate volume": {
"type": "number",
"value": "4.0 mL (post-dialysis)"
},
"Concentration": {
"type": "number",
"value": "2.05 mg/mL"
},
"Total yield": {
"type": "number",
"value": "8.2 mg"
},
"Purity (SDS-PAGE)": {
"type": "number",
"value": "95 %"
}
}
},
"attached_files": [
{
"filename": "akta_chromatogram.pdf",
"type": "pdf",
"size": "342 KB"
},
{
"filename": "sds_page_gel.tif",
"type": "png",
"size": "2.4 MB"
},
{
"filename": "fraction_qc.csv",
"type": "csv",
"size": "14 KB"
}
],
"steps": [
{
"n": 1,
"description": "Inoculate 1 L LB+Kan with 10 mL overnight starter at OD₆₀₀ 0.05, grow 37 °C / 200 rpm",
"duration": "3:30",
"complete": true,
"note": "Reach OD 0.6 at ~3 h"
},
{
"n": 2,
"description": "Induce with 0.5 mM IPTG at OD₆₀₀ 0.7, shift to 16 °C, grow 18 h",
"duration": "18:00",
"complete": true
},
{
"n": 3,
"description": "Harvest by centrifugation 6,000 × g × 15 min, pellet stored -80 °C if not processed same day",
"duration": "0:25",
"complete": true,
"note": "Wet weight 4.2 g"
},
{
"n": 4,
"description": "Resuspend pellet in 50 mL lysis buffer + lysozyme + protease inhibitors",
"duration": "0:20",
"complete": true,
"note": "Lysate volume 60 mL"
},
{
"n": 5,
"description": "Sonicate on ice: 30 s on / 30 s off × 6 cycles, 40% amplitude",
"duration": "0:08",
"complete": true
},
{
"n": 6,
"description": "Clarify at 30,000 × g × 30 min, filter 0.45 µm",
"duration": "0:45",
"complete": true
},
{
"n": 7,
"description": "Equilibrate 5 mL HisTrap HP, load lysate at 1 mL/min, monitor A₂₈₀",
"duration": "1:30",
"complete": true,
"note": "FT collected for SDS-PAGE QC"
},
{
"n": 8,
"description": "Wash steps: 10 CV 20 mM imid, 5 CV 50 mM imid; elute with 5 CV 250 mM imid, collect 1.5 mL fracs",
"duration": "1:20",
"complete": true,
"note": "Peak in fractions E4-E8"
},
{
"n": 9,
"description": "SDS-PAGE QC: lysate / FT / wash / elution fractions; Bradford assay on pooled fractions",
"duration": "2:30",
"complete": true,
"note": "Pool E4-E8; conc 1.8 mg/mL pre-concentration"
},
{
"n": 10,
"description": "Dialyze pooled elution → storage buffer 4 h, concentrate to ~3 mg/mL via Amicon 30 K, archive at -80 °C",
"duration": "5:00",
"complete": true,
"note": "Final: 4 mL × 2.05 mg/mL = 8.2 mg total"
}
],
"spreadsheet": [
[
"Fraction",
"Vol (mL)",
"A₂₈₀",
"Conc (mg/mL)",
"SDS-PAGE",
"Decision"
],
[
"FT",
"60",
"0.42",
"0.18",
"many bands",
"discard"
],
[
"W1 20 mM",
"40",
"0.21",
"0.09",
"few contaminants",
"discard"
],
[
"W2 50 mM",
"25",
"0.08",
"0.03",
"very faint",
"discard"
],
[
"E1",
"1.5",
"0.32",
"0.14",
"target faint",
"discard (front)"
],
[
"E2",
"1.5",
"0.88",
"0.39",
"target + 1 contam",
"pool conditionally"
],
[
"E3",
"1.5",
"2.41",
"1.07",
"clean 72 kDa",
"pool"
],
[
"E4",
"1.5",
"3.05",
"1.36",
"clean 72 kDa",
"pool"
],
[
"E5",
"1.5",
"3.41",
"1.52",
"clean 72 kDa",
"pool"
],
[
"E6",
"1.5",
"2.18",
"0.97",
"clean 72 kDa",
"pool"
],
[
"E7",
"1.5",
"1.04",
"0.46",
"clean 72 kDa",
"pool"
],
[
"E8",
"1.5",
"0.51",
"0.23",
"clean 72 kDa",
"pool"
]
],
"links": {
"experiments": [
{
"ref_id": "EXP-2026-0524",
"title": "BL21(DE3) transformation + clone selection",
"owner": "Y. Nakamura",
"date": "May 24"
},
{
"ref_id": "EXP-2026-0610",
"title": "His₆-MBP-GFP crystallography screen (1024)",
"owner": "Y. Nakamura",
"date": "Jun 10"
}
],
"resources": [
{
"ref_id": "SOP-0623",
"title": "His-tag Protein Purification SOP v2.3",
"owner": "QA Team",
"date": "Jun 2"
},
{
"ref_id": "PLM-0124",
"title": "pET28a-His₆-MBP-GFP construct sequence + map",
"owner": "Plasmid DB",
"date": "Jan 15"
},
{
"ref_id": "AKT-MET",
"title": "ÄKTA HisTrap method file (UNICORN)",
"owner": "Instrument Docs",
"date": "Oct 2"
}
]
},
"compounds": [
{
"name": "HisTrap HP 5 mL column (Cytiva)",
"catalog_lot": "17524802 · Lot 14201842",
"stock_status": "In use"
},
{
"name": "IPTG (Isopropyl β-D-1-thiogalactopyranoside)",
"catalog_lot": "I6758 · Lot SLBS1421",
"stock_status": "In stock"
},
{
"name": "Imidazole (high-purity, BioXtra)",
"catalog_lot": "I2399 · Lot SHBT4821",
"stock_status": "In stock"
},
{
"name": "Lysozyme (egg-white, BioUltra)",
"catalog_lot": "L7651 · Lot SLBP2421",
"stock_status": "In stock"
},
{
"name": "cOmplete EDTA-free protease inhibitor",
"catalog_lot": "11873580001 · Lot 4218",
"stock_status": "In stock"
},
{
"name": "Bradford reagent (Coomassie)",
"catalog_lot": "23200 · Lot UC289421",
"stock_status": "Low"
}
],
"storage": [
{
"name": "Glycerol stocks (BL21-pET28a-MBP-GFP)",
"location": "-80 °C · Freezer A-2 · Drawer 5 · Box GS-2026",
"count": "×6"
},
{
"name": "Purified protein aliquots (50 µL × 2 mg/mL)",
"location": "-80 °C · Freezer A-1 · Drawer 1 · Box MBP-2026-06",
"count": "×40"
},
{
"name": "FPLC chromatogram + SDS-PAGE images",
"location": "Cloud · /preps/2026-06/MBP-GFP-044/",
"count": "×3"
}
],
"permissions": {
"visibility": [
{
"type": "Team",
"name": "Nakamura Lab"
},
{
"type": "Person",
"name": "Y. Nakamura"
}
],
"can_write": [
{
"type": "Person",
"name": "Y. Nakamura"
},
{
"type": "Person",
"name": "Dr. L. Wright"
}
]
}
}