Characteristics of Ni Sepharose^®, Ni Sepharose^® excel, TALON^® Superflow™, and Uncharged IMAC Sepharose^® Products

• Ni Sepharose^® Products
• Stripping, recharging, and cleaning of Ni Sepharose^® products
• Ni Sepharose^® excel/His Mag Sepharose^® excel products
• TALON^® Superflow™ products
  
• Stripping, recharging, and cleaning of TALON^® Superflow™ products
• Uncharged IMAC Sepharose^® products
• Stripping, recharging, and cleaning of IMAC Sepharose^® products

Ni Sepharose^® products

Ni Sepharose^® High Performance is recommended for high-resolution purification of histidine-tagged proteins, providing sharp peaks and concentrated eluate. Ni Sepharose^® 6 Fast Flow is excellent for scaling up and batch purifications. Ni Sepharose^® excel is designed for capture and purification of histidine-tagged proteins secreted into eukaryotic cell culture supernatants. Two magnetic bead formats are also available: His Mag Sepharose^® Ni and His Mag Sepharose^® excel. Table A1.1 summarizes key characteristics of bulk Ni Sepharose^® High Performance and Ni Sepharose^® 6 Fast Flow chromatography media, and Table A1.2 lists the stability of these media under various conditions. Tables A1.3 to A1.11 summarize the characteristics of these same media as prepacked columns, prepacked 96-well plates, and magnetic beads.

Table A1.1Characteristics of Ni Sepharose® High Performance and Ni Sepharose® 6 Fast Flow

Characteristics	Ni Sepharose® High Performance	Ni Sepharose® 6 Fast Flow
Matrix	Highly cross-linked 6% agarose, precharged with Ni2+	Highly cross-linked 6% agarose, precharged with Ni2+
Metal ion capacity	Approx. 15 µmol Ni2+/mL medium	Approx. 15 µmol Ni2+/mL medium
Average particle size	34 µM	90 µM
Dynamic binding capacity1	At least 40 mg (histidine)6-tagged protein/mL medium	Approx. 40 mg (histidine)6-tagged protein/mL medium
Recommended flow velocity2	< 150 cm/h	50–400 cm/h
Compatibility during use	Stable in all commonly used buffers, reducing agents, denaturing agents, and detergents. Table A1.2 for more information.	Stable in all commonly used buffers, reducing agents, denaturing agents, and detergents. Table A1.2 for more information.
Chemical stability3	For 1 wk at 40 °C: 0.01 M HCl, 0.1 M NaOH For 12 h: 1 M NaOH, 70% acetic acid 30 min tested: 30% 2-propanol 1 h tested: 2% SDS	For 1 wk at 40 °C: 0.01 M HCl, 0.1 M NaOH For 12 h: 1 M NaOH, 70% acetic acid 30 min tested: 30% 2-propanol 1 h tested: 2% SDS
pH stability3	Short term (< 2 h) 2–14 Long term (< 1 wk) 3–12	Short term (< 2 h) 2–14 Long term (< 1 wk) 3–12
Storage	20% ethanol	20% ethanol
Storage temperature	4 °C to 30 °C	4 °C to 30 °C

¹ Dynamic binding capacity conditions:

Sample: Column volume: Flow rate: Binding buffer: Elution buffer:

1 mg/mL (histidine)6-tagged pure protein (Mr 43 000) in binding buffer or (histidine)6-tagged protein (Mr 28 000) bound from E. coli extract. Capacity determined at 10% breakthrough. 0.25 mL or 1 mL 0.25 mL/min or 1 mL/min, respectively 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4

Note: Dynamic binding capacity is protein dependent.
² H₂O at room temperature.
³ Ni²⁺-stripped medium.

Table A1.2Compatibility guide: Ni Sepharose^® High Performance, Ni Sepharose^® 6 Fast Flow, and His Mag Sepharose^® Ni are stable toward these compounds at least at the concentrations given

Compound	Concentration
Reducing agents1	5 mM DTE 5 mM DTT 20 mM β-mercaptoethanol 5 mM TCEP (Tris[carboxyethyl] phosphine) 10 mM reduced glutathione
Denaturing agents	8 M urea2 6 M Gua-HCl2
Detergents	2% Tween 20 (nonionic) 2% NP-40 (nonionic) 2% cholate (anionic) 1% CHAPS (zwitterionic)
Other additives	500 mM imidazole 20% ethanol 50% glycerol 100 mM Na2SO4 1.5 M NaCl 1 mM EDTA3 60 mM citrate2
Buffers	50 mM sodium phosphate, pH 7.4 100 mM Tris-HCl, pH 7.4 100 mM Tris-acetate, pH 7.4 100 mM HEPES, pH 7.4 100 mM MOPS, pH 7.4 100 mM sodium acetate, pH 42

¹ Before performing runs with sample/buffers containing reducing reagents, a blank run with binding and elution buffers excluding reducing agents is recommended (see, for example, Manual purification using HisTrap FF crude Kit with a syringe).
² Tested for 1 wk at 40 °C.
³ The strong chelator EDTA has been used successfully in some cases, at 1 mM. Generally, chelating agents should be used with caution (and only in the sample, not in the buffer). Any metal-ion stripping may be counteracted by adding a small excess of MgCl₂ before centrifugation/filtration of the sample. Note that stripping effects may vary with the applied sample volume.

Table A1.3Characteristics of His MultiTrap™ HP and His MultiTrap™ FF

Chromatography media	His MultiTrap™ HP: Ni Sepharose® High Performance His MultiTrap™ FF: Ni Sepharose® 6 Fast Flow; see Table A1.1 for details
Filter plate size1 Filter plate material	127.8 × 85.5 × 30.6 mm Polypropylene and polyethylene
Binding capacity2	His MultiTrap™ HP: Up to 1 mg histidine-tagged protein/well His MultiTrap™ FF: Up to 0.8 mg histidine-tagged protein/well
Reproducibility between wells Volume packed medium/well Number of wells Well volume Max. sample loading volume pH stability3 Storage Storage temperature	+/- 10% 50 µL 96 800 µL 600 µL 2–14 (short term), 3–12 (long term) 20% ethanol 4 °C to 30 °C

¹ According to ANSI/SBS 1-2004, 3-2004, and 4-2004 standards (ANSI = American National Standards Institute and SBS = Society for Biomolecular Screening).
² Protein binding capacity is protein dependent.
³ Ni²⁺-stripped medium.

Table A1.4Characteristics of His SpinTrap

Chromatography medium Average particle size Bed volume Column material Protein binding capacity1 Compatibility during use	Ni Sepharose® High Performance; Table A1.1 for details 34 µM 100 µL Polypropylene barrel and polyethylene frits Approx. 0.75 mg histidine-tagged protein/column Stable in all commonly used buffers, reducing agents, denaturants, and detergents. Table A1.2 for more information.
Storage Storage temperature	0.15% Kathon™ CG 4 °C to 30 °C

¹ Protein binding capacity is protein dependent.

Table A1.5Characteristics of HisTrap HP and HisTrap FF

Chromatography media	HisTrap HP: Ni Sepharose® High Performance HisTrap FF: Ni Sepharose® 6 Fast Flow; Table A1.1 for details
Column volume Column dimensions	1 mL and 5 mL 0.7 × 2.5 cm (1 mL); 1.6 × 2.5 cm (5 mL)
Dynamic binding capacity1	HisTrap HP: At least 40 mg histidine-tagged protein/mL medium HisTrap FF: Approx. 40 mg histidine-tagged protein/mL medium
Recommended flow rate	1 mL/min (1 mL); 5 mL/min (5 mL)
Max. flow rate2 Max. pressure2 pH stability3	4 mL/min (1 mL); 20 mL/min (5 mL) 0.3 MPa, 3 bar 2–14 (short term), 3–12 (long term)
Compatibility	Stable in all commonly used buffers, reducing agents, denaturants, and detergents. Table A1.2 for more information.
Chemical stability3	For 1 wk at 40 °C: 0.01 M HCl, 0.1 M NaOH For 12 h: 1 M NaOH, 70% acetic acid 30 min tested: 30% 2-propanol 1 h tested: 2% SDS
Storage Storage temperature	20% ethanol 4 °C to 30 °C

¹ Dynamic binding capacity conditions:

Sample: Column volume: Flow rate: Binding buffer: Elution buffer:

1 mg/mL (histidine)6-tagged pure protein (Mr 43 000) in binding buffer or (histidine)6-tagged protein (Mr 28 000) bound from E. coli extract. Capacity determined at 10% breakthrough. 0.25 mL or 1 mL 0.25 mL/min or 1 mL/min, respectively 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4

Note: Dynamic binding capacity is protein dependent.
² H₂O at room temperature.
³ Ni²⁺-stripped medium.

Table A1.6Characteristics of HisTrap FF crude

Chromatography medium Average particle size Column volume Column dimensions Dynamic binding capacity1 Recommended flow rate2 Max. pressure2	Ni Sepharose® 6 Fast Flow; Table A1.1 for details 90 µM 1 mL and 5 mL 0.7 × 2.5 cm (1 mL); 1.6 × 2.5 cm (5 mL) Approx. 40 mg histidine-tagged protein/mL medium 1 mL/min (1 mL); 5 mL/min (5 mL) 3 bar (0.3 MPa, 42 psi)
Compatibility during use	Stable in all commonly used buffers, reducing agents, denaturing agents, and detergents. Table A1.2 for more information.
Chemical stability3	For 1 wk at 40 °C: 0.01 M HCl, 0.1 M NaOH For 12 h: 1 M NaOH, 70% acetic acid 30 min tested: 30% 2-propanol 1 h tested: 2% SDS
pH stability3 Storage Storage temperature	2–14 (short term), 3–12 (long term) 20% ethanol 4 °C to 30 °C

¹ Dynamic binding capacity conditions:

Sample:	1 mg/mL (histidine)6-tagged pure protein (Mr 43 000) in binding buffer or (histidine)6-tagged protein (Mr 28 000) bound from E. coli extract. Capacity determined at 10% breakthrough.
Column volume: Flow rate: Binding buffer: Elution buffer:	0.25 mL or 1 mL 0.25 mL/min or 1 mL/min, respectively 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4

Note: Dynamic binding capacity is protein dependent.
² H₂O at room temperature.
³ Ni²⁺-stripped medium.

Table A1.7Characteristics and contents of HisTrap FF crude Kit

Sample:	1 mg/mL (histidine)6-tagged pure protein (Mr 43 000) in binding buffer or (histidine)6-tagged protein  (Mr 28 000) bound from E. coli extract. Capacity determined at 10% breakthrough.
Column volume: Flow rate: Binding buffer: Elution buffer:	0.25 mL or 1 mL 0.25 mL/min or 1 mL/min, respectively 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4

¹Table A1.6 for the characteristics of HisTrap FF crude columns.

Table A1.8Characteristics of His GraviTrap

Chromatography medium Average particle size Bed volume Column material Protein binding capacity1	Ni Sepharose® 6 Fast Flow; Table A1.1 for details 90 µM 1 mL Polypropylene barrel, polyethylene frits Approx. 40 mg histidine-tagged protein/column
Compatibility during use	Stable in all commonly used buffers, reducing agents, denaturing agents and detergents. Table A1.2 for more information.
Chemical stability2	For 1 wk at 40 °C: 0.01 M HCl, 0.1 M NaOH For 12 h: 1 M NaOH, 70% acetic acid 30 min tested: 30% 2-propanol 1 h tested: 2% SDS
Storage Storage temperature	20% ethanol 4 °C to 30 °C

¹ Protein binding capacity is protein dependent.
² Ni²⁺-stripped medium.

Table A1.9Characteristics of HiScreen Ni FF

Medium Matrix Particle size, d50v1 Dynamic binding capacity2 Metal ion capacity Recommended flow velocity3 Maximum flow velocity3	Ni Sepharose® 6 Fast Flow; Table A1.1 for details Highly cross-linked 6% agarose 90 µM Approx. 40 mg (histidine)6-tagged protein/mL medium Approx. 15 µmol Ni2+/mL medium 30 to 300 cm/h 450 cm/h
Maximum pressure over the packed bed  during operation, ∆p4	0.15 MPa, 1.5 bar, 22 psi
HiScreen column hardware pressure limit	0.5 MPa, 5 bar, 73 psi
pH stability5 (for medium without metal ion) -          short term (at least 2 h) -          long term (≤ 1 wk)	2–14 3–12
Compatibility during use	Stable in all commonly used buffers, reducing agents, denaturants, and detergents. Table A1.2 for more information.
Chemical stability (for medium without metal ion)	12 h tested: 1 M NaOH, 70% acetic acid 1 h tested: 2% SDS 30 min tested: 30% 2-propanol
Avoid in buffers Storage	Chelating agents, e.g., EDTA6, EGTA, citrate 4 °C to 30 °C in 20% ethanol

¹ d_50v is the average particle size of the cumulative volume distribution.
² Dynamic binding capacity conditions:

Samples:	1 mg/mL (histidine)6-tagged pure protein (Mr 43 000) in binding buffer (QB, 10% determination) or histidine)6-tagged protein (Mr 28 000) bound from E. coli extract.
Column volumes: Flow rates: Binding buffer: Elution buffer:	0.25 or 1 mL 0.25 or 1 mL/min, respectively 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, pH 7.4 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4

Note: Dynamic binding capacity is protein dependent.
³ H₂O at room temperature.
⁴ Many chromatography systems are equipped with pressure gauges to measure the pressure at a particular point in the system, usually just after the pumps. The pressure measured here is the sum of the pre-column pressure, the pressure drop over the medium bed, and the post-column pressure. It is always higher than the pressure drop over the bed alone. We recommend keeping the pressure drop over the bed below 1.5 bar. Setting the upper limit of your pressure gauge to 1.5 bar will ensure the pump shuts down before the medium is overpressured. If necessary, post-column pressure of up to 3.5 bar can be added to the limit without exceeding the column hardware limit. To determine post-column pressure, proceed as follows:
To avoid breaking the column, the post-column pressure must never exceed 3.5 bar.

1. Connect a piece of tubing in place of the column.
2. Run the pump at the maximum flow you intend to use for chromatography. Use a buffer with the same viscosity as you intend to use for chromatography. Note the back pressure as total pressure.
3. Disconnect the tubing and run at the same flow rate used in step 2. Note this back pressure as pre-column pressure.
4. Calculate the post-column pressure as total pressure minus pre-column pressure. If the post-column pressure is higher than 3.5 bar, take steps to reduce it (shorten tubing, clear clogged tubing, or change flow restrictors), and perform steps 1 to 4 again until the post-column pressure is below 3.5 bar. When the post-column pressure is satisfactory, add the post-column pressure to 1.5 bar and set this as the upper pressure limit on the chromatography system.

⁵ Short term pH: pH interval where the medium can be subjected to cleaning- or sanitization-in-place without significant change in function.
Long term pH: pH interval where the medium can be operated without significant change in function.
⁶ The strong chelator EDTA has been used successfully in some cases at 1 mM. Generally, chelating agents should be used with caution (and only in the sample, not in the buffer). Any metal-ion stripping may be counteracted by adding a small excess of MgCl₂ before centrifugation/filtration of the sample. Note that stripping effects may vary with the applied sample volume.

Table A1.10Characteristics of HisPrep FF 16/10

Chromatography medium Column volume Column dimensions Dynamic binding capacity1 Recommended flow rate2	Ni Sepharose® 6 Fast Flow; Table A1.1 for details 20 mL 1.6 × 10 cm Approx. 40 mg histidine-tagged protein/mL medium 2–10 mL/min (60–300 cm/h)
Max. flow rate2 Max. pressure over the packed bed during operation2	10 mL/min (300 cm/h) 1.5 bar (0.15 MPa, 22 psi)
Column hardware pressure limit	5 bar (0.5 MPa, 73 psi)
Compatibility during use	Stable in all commonly used buffers, reducing agents, denaturing agents and detergents. Table A1.2 for more information.
Chemical stability3	For 1 wk at 40 °C: 0.01 M HCl, 0.1 M NaOH For 12 h: 1 M NaOH, 70% acetic acid 30 min tested: 30% 2-propanol 1 h tested: 2% SDS
Storage Storage temperature	20% ethanol 4 °C to 30 °C

¹ Dynamic binding capacity conditions:

Sample:	1 mg/mL (histidine)6-tagged pure protein (Mr 43 000) in binding buffer or (histidine)6-tagged protein (Mr 28 000) bound from E. coli extract. Capacity determined at 10% breakthrough.
Column volume: Flow rate: Binding buffer: Elution buffer:	0.25 mL or 1 mL 0.25 mL/min or 1 mL/min, respectively 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4

Note: Dynamic binding capacity is protein dependent.
² H₂O at room temperature.
³ Ni²⁺-stripped medium.

Table A1.11Characteristics of His Mag Sepharose® Ni

Matrix Metal ion capacity Binding capacity1	Highly cross-linked spherical agarose (Sepharose) including magnetite Approx. 21 µmol Ni2+/mL medium Approx. 50 mg histidine-tagged protein/mL sedimented medium (~500 µg/purification run)
Particle size Working temperature Compatibility during use	37 to 100 µM Room temperature and 4 °C Stable in all commonly used buffers, reducing agents, denaturants such as 6 M Gua-HCl and 8 M urea, and a range of other additives. Table A1.2 for further information.
Storage solution Storage temperature	20% ethanol, 5% medium slurry Room temperature

¹ The capacity was determined using 5 mM imidazole in sample and binding buffer. Note that binding capacity is sample dependent.

Stripping, recharging, and cleaning of Ni Sepharose^® products

Stripping and Recharging

Ni Sepharose High Performance and Ni Sepharose 6 Fast Flow do not have to be stripped and recharged between each purification if the same protein is to be purified. It may be sufficient to strip and recharge it after approximately two to five purifications, depending on the specific sample, sample pretreatment, sample volume, etc.

Stripping buffer: 20 mM sodium phosphate, 500 mM NaCl, 50 mM EDTA, pH 7.4

1. Strip the chromatography medium by washing with at least 5 to 10 column volumes of stripping buffer.
2. Wash with at least 5 to 10 column volumes of binding buffer.
3. Immediately wash with 5 to 10 column volumes of distilled water.
4. Recharge the water-washed column by loading 0.5 column volumes of 0.1 M NiSO₄ in distilled water onto the column.
5. Wash with 5 column volumes of distilled water, and 5 column volumes of binding buffer (to adjust pH) before storage in 20% ethanol. Salts of other metals, chlorides, or sulfates may also be used.

It is important to wash with binding buffer as the last step to obtain the correct pH before storage.

Washing with buffer before applying the metal ion solution may cause unwanted precipitation.

Cleaning-in-place

When an increase in back pressure is seen, the chromatography medium should be cleaned. Before cleaning, strip off metal ions using the recommended procedure described above. The stripped medium can be cleaned by the following methods:

To remove ionically bound protein:

1. Wash with several column volumes of 1.5 M NaCl.
2. Immediately wash with approximately 10 column volumes of distilled water.

To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins:

1. Wash the column with 1 M NaOH, contact time usually 1 to 2 h (12 h or more for endotoxin removal).
2. Immediately wash with approximately 10 column volumes of binding buffer, followed by 5 to 10 column volumes of distilled water.

To remove hydrophobically bound proteins, lipoproteins, and lipids:

1. Wash with 5 to 10 column volumes of 30% isopropanol for about 15 to 20 min.
2. Immediately wash with approximately 10 column volumes of distilled water.
   
   2a. Alternatively, wash with 2 column volumes of detergent in a basic or acidic solution. Use, for example, 0.1 to 0.5% nonionic detergent in 0.1 M acetic acid, contact time 1 to 2 h. After treatment, always remove residual detergent by washing with at least 5 column volumes of 70% ethanol. Then wash with approximately 10 column volumes of distilled water.

Reversed flow may improve the efficiency of the cleaning-in-place procedure. After cleaning, store in 20% ethanol (wash with 5 column volumes) or recharge with Ni²⁺ prior to storage in ethanol.

Ni Sepharose^® excel/His Mag Sepharose^® excel products

Ni Sepharose^® excel and His Mag Sepharose^® excel are designed for capture and purification of histidine-tagged proteins secreted into eukaryotic cell culture supernatants. Nickel ions are very strongly bound to both chromatography media, enabling direct loading of large sample volumes without removing agents that normally would cause metal ion stripping. His Mag Sepharose^® excel is magnetic beads designed for simple and efficient purification and screening. Ni Sepharose^® excel is available for all scales of work from convenient, prepacked HisTrap excel columns to bulk quantities.
Table A1.12 summarizes key characteristics of both media, and Table A1.13 lists the stability of Ni Sepharose^® excel under various conditions. Table A1.14 summarizes the characteristics of Ni Sepharose^® excel prepacked as a HisTrap excel column.

Table A1.12Characteristics of His Mag Sepharose® excel and Ni Sepharose® excel

Product	His Mag Sepharose® excel	Ni Sepharose® excel
Matrix	Highly cross-linked spherical agarose including magnetite	Highly cross-linked spherical agarose
Precharged ion Average particle size Binding capacity1	Nickel 63 µM At least 10 mg (histidine)6-tagged protein/mL sedimented medium	Nickel 90 µM At least 10 mg (histidine)6-tagged protein/mL sedimented medium
Maximum flow velocity2,3	N/A	600 cm/h
pH stability4	Working range: 3 to 12 Cleaning-in-place: N/A	Working range: 3 to 12 Cleaning-in-place: 2 to 14
Storage	4 °C to 30 °C in 20% ethanol	4 °C to 30 °C in 20% ethanol

¹ Binding capacity is sample dependent.
² H₂O at room temperature.
³ Optimal flow velocity during binding is sample dependent. During column wash and elution, a flow velocity of 150 cm/h is recommended.
⁴ Working range: pH interval where the medium can be operated without significant change in function.
Cleaning-in-place: pH interval where the medium can be subjected to cleaning-in-place without significant change in function.

Table A1.13Chemical stability1 of Ni Sepharose® excel

Substance	Duration of test
0.01 M HCl and 0.01 M NaOH 10 mM EDTA, 1 M NaOH, 5 mM DTT, 5 mM TCEP, 20 mM β-mercaptoethanol, and 6 M Gua-HCl	1 wk 24 h
500 mM imidazole and 100 mM EDTA 30% 2-propanol	2 h 20 min

¹ Chemical stability was tested by incubating the medium in the listed solutions at room temperature, and thereafter measuring either the nickel leakage or the protein binding capacity.

Table A1.14Main characteristics of HisTrap excel

Column Medium Column volumes Column dimensions	HisTrap excel Ni Sepharose® excel 1 mL and 5 mL 0.7 × 2.5 cm (1 mL column) 1.6 × 2.5 cm (5 mL column)
Recommended flow rates1,2	1 to 4 mL/min (1 mL column) 5 to 20 mL/min (5 mL column)
Maximum flow rates1	4 mL/min (1 mL column) 20 mL/min (5 mL column)
Column hardware pressure limit	5 bar (0.5 MPa)

¹ H₂O at room temperature. Maximum flow rate will be lower when using buffers or samples with high viscosity or when performing purification at low temperature.
² Optimal flow rate during binding is sample dependent. During column wash and elution, a flow rate of 1 mL/min and 5 mL/min is recommended for 1 mL and 5 mL columns, respectively.
Note: The maximum pressure the packed bed can withstand depends on the chromatography medium characteristics and sample/liquid viscosity. The value measured on the chromatography system used also depends on the tubing used to connect the column.

TALON^® Superflow™ products

TALON^® Superflow™ is a cobalt-based immobilized metal ion affinity chromatography medium (IMAC) offering enhanced selectivity for histidine-tagged proteins compared with nickel-charged chromatography media. TALON^® Superflow™ is available for all scales of work from 96-well plates to convenient, prepacked columns to bulk quantities, enabling different throughput and scales from screening in low microgram scale to milligram preparative purification of histidine-tagged proteins.

Table A1.15 summarizes key characteristics of bulk TALON^® Superflow™ medium, and Table A1.16 lists the stability of the medium under various conditions. Tables A1.17 to A1.18 summarize the characteristics of these same media as prepacked columns and as prepacked 96-well plates.

Table A1.15Characteristics of TALON^® Superflow

Matrix Precharged ion Particle size distribution Binding capacity1 Maximum flow velocity2	Cross-linked agarose, 6% Cobalt 60 µM to 160 µM up to 20 mg histidine-tagged protein/mL medium 2000 cm/h
pH stability3,4       Short term (2 h)       Long term (1 wk)	2 to 14 3 to 12
Storage Compatibility during use	20% ethanol at 4 °C to 8 °C Stable in all commonly used buffers, denaturants, and detergents (Table A1.16)

¹ The binding capacity for individual proteins may vary.
² H₂O in a 0.75 × 10 cm (i.d. × H) column.
³ Co²⁺-stripped medium.
⁴ Below pH 4, metal ions will be stripped off the medium, and therefore neutral to slightly alkaline pH (pH 7 to 8) is recommended.

Table A1.16Compatible reagents for TALON® Superflow1,2

Reagent	Acceptable concentration
β-Mercaptoethanol3 CHAPS, SDS, sarcosyl4 Ethanol5 Ethylene glycol HEPES Glycerol Gua-HCl Imidazole6 KCl MES MOPS NaCl NP-40 Tris7 Tween-20 Urea	10 mM (with caution) 1% (with caution) 30% 30% 50 mM 20% 6 M ≤ 500 mM at pH 7.0 to 8.0, for elution 500 mM 20 mM 50 mM 1.0 M 1% 50 mM < 1% 8 M

¹ Data provided by Clontech Laboratories, Inc.
² EDTA and other chelators, such as EGTA, will strip Co²⁺ ions from the medium; EDTA may be used, but must be removed prior to sample application. Strong reducing agents should also be avoided (e.g., DTT, DTE, and TCEP) because they interfere with Co²⁺ ions binding to the medium.
³ Use TALON^® Superflow™ immediately after equilibrating with buffers containing β-mercaptoethanol, otherwise the medium will change color. Do not store the medium in buffers containing β-mercaptoethanol.
⁴ Ionic detergents like CHAPS, SDS, and sarcosyl are compatible up to 1%. However, due to their charged nature, you should anticipate interference with binding.
⁵ Ethanol may precipitate proteins, causing low yields and column clogging.
⁶ Imidazole at concentrations higher than 5 to 10 mM may cause lower yields of histidine-tagged proteins because it competes with the histidine side chains (imidazole groups) for binding to the immobilized metal ions.
⁷ Tris coordinates weakly with metal ions, causing a decrease in capacity.

Table A1.17Characteristics of His GraviTrap TALON®, His SpinTrap TALON®, and His MultiTrap TALON®

Column and 96-well plate material	Polypropylene barrel and plate, polyethylene frits
Protein binding capacity1    His GraviTrap TALON®    His SpinTrap TALON®    His MultiTrap TALON®	Up to 15 mg histidine-tagged protein/column Up to 1 mg histidine-tagged protein/column Up to 1 mg histidine-tagged protein/well
Bed volume in columns/wells    His GraviTrap TALON®    His SpinTrap TALON®    His MultiTrap TALON®	1 mL/column 100 µL/column 50 µL/well (500 µL of 10% slurry)
Total volume in columns/wells    His GraviTrap TALON®    His SpinTrap TALON®    His MultiTrap TALON®	13.5 mL 1000 µL 800 µL
Reproducibility, His MultiTrap TALON®, column-to column, plate-to-plate, and well-to-well	±10%
Filter plate size of His MultiTrap TALON®	127.8 × 85.5 × 30.6 mm according to ANSI/SBS 1-2004, 3-2004 and 4-2004 standards
Number of wells Avoid in buffers	96 Chelating agents, e.g. EDTA, EGTA, citrate and DTT, DTE, and TCEP

¹ The binding capacity for individual proteins may vary.

Table A1.18Characteristics of HiTrap TALON® crude

Column volume	1 mL and 5 mL
Column dimensions, i.d. × H	0.7 × 2.5 cm (1 mL column) 1.6 × 2.5 cm (5 mL column)
Recommended flow rate1	1 mL/min (1 mL column) 5 mL/min (5 mL column)
Maximum flow rate1	4 mL/min (1 mL column) 20 mL/min (5 mL column)
Column hardware pressure limit2 Chromatography medium	0.5 MPa, 5 bar TALON® Superflow™ (Table A1.15)

¹ H₂O at room temperature.
² The pressure over the packed bed varies depending on a range of parameters such as the characteristics of the chromatography medium and the column tubing used.

Stripping, recharging, and cleaning of TALON^® Superflow™ products

TALON^® Superflow™ and HiTrap TALON^® crude do not have to be stripped and recharged between each purification if the same protein is to be purified. It may be sufficient to strip and recharge it after approximately two to five purifications, depending on the specific sample, sample pretreatment, sample volume, etc.

Purification of histidine-tagged proteins using imidazole gradients will cause TALON^® Superflow™ to take on a dark purplish color. Washing the medium with 5 to 10 bed/column volumes of 20 mM MES Buffer (pH 5.0) will restore the normal pink color and bring the absorbance at 280 nm back to the original baseline level. After equilibrating with binding buffer (50 mM sodium phosphate, 300 mM NaCl, pH 7.4), the medium/column is ready for reuse.

Stripping and recharging

Stripping buffer: 200 mM EDTA, pH 7.0

1. Strip the TALON^® Superflow™/HiTrap TALON^® crude of cobalt ions by washing with 10 bed/column volumes of stripping buffer.
2. Wash excess EDTA from the medium with an additional 10 bed/column volumes of distilled water.
3. Charge the chromatography medium with 10 bed/column volumes of 50 mM CoCl₂ solution.
4. Wash with 7 bed/column volumes of distilled water followed by 3 bed/column volumes of 300 mM NaCl and by 3 bed/column volumes of distilled water to remove excess cobalt metal ions.
5. Equilibrate with 10 bed/column volumes of binding buffer.

It is important to wash with binding buffer as the last step to obtain the correct pH before storage.

Uncharged IMAC Sepharose^® products

IMAC Sepharose^® High Performance is recommended for high-resolution purifications, providing sharp peaks and concentrated eluate. IMAC Sepharose^® 6 Fast Flow is excellent for scaling up.
Table A1.19 summarizes key characteristics of IMAC Sepharose^® media, and Table A1.20 lists the stability of the media under various conditions. Tables A1.21 to A1.23 summarize the characteristics of the media as prepacked columns.

Table A1.19Characteristics of IMAC Sepharose® High Performance and IMAC Sepharose® 6 Fast Flow

Characteristics	IMAC Sepharose® High Performance	IMAC Sepharose® 6 Fast Flow
Matrix	Highly cross-linked 6% spherical agarose	Highly cross-linked 6% spherical agarose
Metal ion capacity Average particle size	Approx. 15 µmol Ni2+/mL medium 34 µM	Approx. 15 µmol Ni2+/mL medium 90 µM
Dynamic binding capacity1	At least 40 mg (histidine)6-tagged protein/mL medium (Ni2+-charged)	Histidine-tagged protein: Approx. 40 mg (histidine)6-tagged protein/mL medium (Ni2+-charged) Untagged protein: Approx. 25 mg/mL medium (Cu2+-charged); approx. 15 mg/mL  medium (Zn2+ or Ni2+-charged).
Recommended flow velocity2	< 150 cm/h	150 cm/h
Compatibility during use	Stable in all commonly used buffers, reducing agents, denaturing agents, and detergents. See Table A1.20 for more information.	Stable in all commonly used buffers, reducing agents, denaturing agents, and detergents. See Table A1.20 for more information.
Chemical stability3	For 1 wk at 40 °C: 0.01 M HCl, 0.1 M NaOH For 12 h: 1 M NaOH, 70% acetic acid 1 h tested: 2% SDS 30 min tested: 30% 2-propanol	For 1 wk at 40 °C: 0.01 M HCl, 0.1 M NaOH For 12 h: 1 M NaOH, 70% acetic acid 1 h tested: 2% SDS 30 min tested: 30% 2-propanol
pH stability3	Short term (< 2 h): 2–14 Long term (< 1 wk): 3–12	Short term (< 2 h): 2–14 Long term (< 1 wk): 3–12
Storage Storage temperature	20% ethanol 4 °C to 30 °C	20% ethanol 4 °C to 30 °C

¹ Conditions for determining dynamic binding capacity:

Samples:	(Histidine)6-tagged proteins: Capacity data were obtained for a protein (Mr 28 000) bound from an E. coli extract, and a pure protein (Mr 43 000) applied at 1 mg/mL in binding buffer; capacity at 10% breakthrough. Untagged protein (IMAC Sepharose® 6 Fast Flow only): Capacities determined at 10% breakthrough for human apotransferrin applied at 1 mg/mL in binding buffer.
Column volume:	0.25 mL or 1 mL
Flow rate: Binding buffer: Elution buffer:	0.25 mL/min or 1 mL/min, respectively 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole (1 mM for untagged protein, IMAC Sepharose® 6 Fast Flow only), pH 7.4 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole (50 mM for untagged protein, IMAC Sepharose® 6 Fast Flow only), pH 7.4

Note: Dynamic binding capacity is metal ion and protein dependent.
² H₂O at room temperature.
³ Uncharged medium only. See Table A1.20 for more information.

Table A1.20Compatibility guide: IMAC Sepharose® High Performance and IMAC Sepharose® 6 Fast Flow are stable toward these compounds at least at the concentrations given

Compound	Concentration
Reducing agents1	5 mM DTE 5 mM DTT 20 mM β-mercaptoethanol 5 mM TCEP 10 mM reduced glutathione
Denaturing agents	8 M urea2 6 M Gua-HCl2
Detergents	2% Tween 20 (nonionic) 2% NP-40 (nonionic) 2% cholate (anionic) 1% CHAPS (zwitterionic)
Other additives	500 mM imidazole 20% ethanol 50% glycerol 100 mM Na2SO4 1.5 M NaCl 1 mM EDTA3 60 mM citrate2
Buffers	50 mM sodium phosphate, pH 7.4 100 mM Tris-HCl, pH 7.4 100 mM Tris-acetate, pH 7.4 100 mM HEPES, pH 7.4 100 mM MOPS, pH 7.4 100 mM sodium acetate, pH 42

¹ Before performing runs with sample/buffers containing reducing reagents, a blank run with binding and elution buffers excluding reducing agents is recommended (see, for example, Blank run in Purification using IMAC Sepharose^® High Performance, Chapter 3).
² Tested for 1 wk at 40 °C.
³ The strong chelator EDTA has been used successfully in some cases, at 1 mM. Generally, chelating agents should be used with caution (and only in the sample, not the buffer). Any metal-ion stripping may be counteracted by adding a small excess of MgCl₂ before centrifugation/filtration of the sample. Note that stripping effects may vary with the applied sample volume.

Table A1.21Characteristics of HiTrap IMAC HP and HiTrap IMAC FF

Chromatography media	HiTrap IMAC HP: IMAC Sepharose® High Performance HiTrap IMAC FF: IMAC Sepharose® 6 Fast Flow
Column volume Dynamic binding capacity1	1 mL or 5 mL At least 40 mg histidine-tagged protein/mL medium when charged with Ni2+. For untagged proteins, HiTrap FF can bind approx. 25 mg/mL medium charged with Cu2+ or approx. 15 mg/mL medium charged with Zn2+ or Ni2+.
Column dimensions Recommended flow rate Max flow rate2 Max. back pressure2 pH stability3 Compatibility during use	0.7 × 2.5 cm (1 mL); 1.6 × 2.5 cm (5 mL) 1 mL/min (1 mL); 5 mL/min (5 mL) 4 mL/min (1 mL); 20 mL/min (5 mL) 0.3 MPa, 3 bar 2–14 (short term), 3–12 (long term) Stable in all commonly used buffers, reducing agents, denaturants, and detergents. Table A1.20 for more information.
Chemical stability3	For 1 wk at 40 °C: 0.01 M HCl, 0.1 M NaOH For 12 h: 1 M NaOH, 70% acetic acid 30 min tested: 30% 2-propanol 1 h tested: 2% SDS
Storage Storage temperature	20% ethanol 4 °C to 30 °C

¹ Conditions for determining dynamic binding capacity:

Samples:	(Histidine)6-tagged proteins: Capacity data were obtained for a protein (Mr 28 000) bound from an E. coli extract, and a pure protein (Mr 43 000) applied at 1 mg/mL in binding buffer; capacity at 10% breakthrough. Untagged protein (IMAC Sepharose® 6 Fast Flow only): Capacities determined at 10% breakthrough for human apotransferrin applied at 1 mg/mL in binding buffer.
Column volume:	0.25 mL or 1 mL
Flow rate: Binding buffer: Elution buffer:	0.25 mL/min or 1 mL/min, respectively 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole (1 mM for untagged protein, IMAC Sepharose® 6 Fast Flow only), pH 7.4 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole (50 mM for untagged protein,IMAC Sepharose® 6 Fast Flow only), pH 7.4.

Note: Dynamic binding capacity is metal ion and protein dependent.
² H₂O at room temperature.
³ Uncharged medium only. See Table A1.20 for more information.

Table A1.22Characteristics of HiScreen IMAC FF

Medium Matrix Particle size, d50v1	IMAC Sepharose® 6 Fast Flow Highly cross-linked 6% agarose 90 µM
Dynamic binding capacity2	Approx. 40 mg (histidine)6-tagged protein/mL medium Untagged protein: Approx. 25 mg/mL medium (Cu2+ charged), or approx. 15 mg/mL medium (Zn2+ or Ni2+ charged).
Metal ion capacity Recommended fluid velocity3 Maximum fluid velocity3 Maximum pressure over the packed bed during operation, ∆p4	Approx. 15 µmol Ni2+/mL medium 30 to 300 cm/h 600 cm/h 0.15 MPa, 1.5 bar, 22 psi
HiScreen column hardware pressure limit	0.5 MPa, 5 bar, 73 psi
pH stability5 (for medium without metal ion) -          short term (at least 2 h) -          long term (≤ 1 week)	2–14 3–12
Compatibility during use	Table A1.20
Chemical stability (for medium without metal ion)	Tested for 12 h: 1 M NaOH, 70% acetic acid Tested for 1 h: 2% SDS Tested for 30 min: 30% 2-propanol
Avoid in buffers Storage	Chelating agents, e.g., EDTA, EGTA, citrate6 4 °C to 30 °C in 20% ethanol

¹ d_50v is the average particle size of the cumulative volume distribution.
² Dynamic binding capacity conditions:

Samples:	1 mg/mL (histidine)6-tagged pure protein (Mr 43 000) in binding buffer (QB, 10% determination) or histidine)6-tagged protein (Mr 28 000) bound from E. coli extract.
Column volumes:	0.25 or 1 mL
Flow rates: Binding buffer: Elution buffer:	0.25 or 1 mL/min, respectively 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, pH 7.4 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4

Note: Dynamic binding capacity is protein dependent.
³ Water at room temperature.
⁴ Many chromatography systems are equipped with pressure gauges to measure the pressure at a particular point in the system, usually just after the pumps. The pressure measured here is the sum of the pre-column pressure, the pressure drop over the medium bed, and the post-column pressure. It is always higher than the pressure drop over the bed alone. We recommend keeping the pressure drop over the bed below 1.5 bar. Setting the upper limit of your pressure gauge to 1.5 bar will ensure the pump shuts down before the medium is overpressured. If necessary, post-column pressure of up to 3.5 bar can be added to the limit without exceeding the column hardware limit. To determine post-column pressure, proceed as follows:
To avoid breaking the column, the post-column pressure must never exceed 3.5 bar.

1. Connect a piece of tubing in place of the column.
2. Run the pump at the maximum flow you intend to use for chromatography. Use a buffer with the same viscosity as you intend to use for chromatography. Note the back pressure as total pressure.
3. Disconnect the tubing and run at the same flow rate used in step 2. Note this back pressure as pre-column pressure.
4. Calculate the post-column pressure as total pressure minus pre-column pressure. If the post-column pressure is higher than 3.5 bar, take steps to reduce it (shorten tubing, clear clogged tubing, or change flow restrictors), and perform steps 1 to 4 again until the post-column pressure is below 3.5 bar. When the post-column pressure is satisfactory, add the post-column pressure to 1.5 bar and set this as the upper pressure limit on the chromatography system.

⁵ Short term pH: pH interval where the medium can be subjected to cleaning- or sanitization-in-place without significant change in function.
Long term pH: pH interval where the medium can be operated without significant change in function.
⁶ The strong chelator EDTA has been used successfully in some cases at 1 mM. Generally, chelating agents should be used with caution (and only in the sample, not in the buffer). Any metal-ion stripping may be counteracted by adding a small excess of MgCl₂ before centrifugation/filtration of the sample. Note that stripping effects may vary with the applied sample volume.

Table A1.23Characteristics of HiPrep IMAC FF 16/10

Chromatography medium Column volume Column dimensions Dynamic binding capacity1	IMAC Sepharose® 6 Fast Flow 20 mL 1.6 × 10 cm Approx. 40 mg histidine-tagged protein/mL medium when charged with Ni2+. For untagged proteins, HiTrap FF binds approx. 25 mg/mL medium charged with Cu2+ or approx. 15 mg/mL medium charged with Zn2+ or Ni2+.
Recommended flow rate2 Max. flow rate2 Max. pressure over the packed bed during operation2	2–10 mL/min (60–300 cm/h) 10 mL/min (300 cm/h) 0.15 MPa, 1.5 bar
Column hardware pressure limit Compatibility during use	0.5 MPa, 5 bar Stable in all commonly used buffers, reducing agents, denaturants, and detergents. Table A1.20 for more information.
Chemical stability3	Tested for 1 wk at 40 °C: 0.01 M HCl, 0.1 M NaOH Tested for 12 h: 1 M NaOH, 70% acetic acid Tested for 1 h: 2% SDS Tested for 30 min: 30% 2-propanol
Storage Storage temperature	20% ethanol 4 °C to 30 °C

¹ Conditions for determining dynamic binding capacity:

Samples:	(Histidine)6-tagged proteins: Capacity data were obtained for a protein (Mr 28 000) bound from an E. coli extract, and a pure protein (Mr 43 000) applied at 1 mg/mL in binding buffer; capacity at 10% breakthrough. Untagged protein: Capacities determined at 10% breakthrough for human apotransferrin applied at 1 mg/mL in binding buffer.
Column volume:	0.25 or 1 mL
Flow rate: Binding buffer: Elution buffer:	0.25 or 1 mL/min, respectively 20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, (1 mM for untagged protein) pH 7.4 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, (50 mM for untagged protein) pH 7.4

Note: Dynamic binding capacity is metal ion and protein dependent.
² H₂O at room temperature.
³ Uncharged medium only. See Table A1.20 for more information.

Stripping, recharging, and cleaning of IMAC Sepharose^® products

IMAC Sepharose High Performance and IMAC Sepharose 6 Fast Flow do not have to be stripped and recharged between each purification if the same protein is to be purified. It may be sufficient to strip and recharge medium after approximately two to five purifications, depending on the specific sample, sample pretreatment, sample volume, etc.

Stripping and recharging

Stripping buffer: 20 mM sodium phosphate, 500 mM NaCl, 50 mM EDTA, pH 7.4

1. Strip the chromatography medium by washing with at least 5 to 10 column volumes of stripping buffer.
2. Wash with at least 5 to 10 column volumes of binding buffer; see Blank run in Purification using IMAC Sepharose High Performance, Chapter 3 (link to your Google Drive).
3. Immediately wash with 5 to 10 column volumes of distilled water.
4. Prepare a 0.1 M solution of the chosen metal ion in distilled water. Salts of chlorides, sulfates, etc., can be used: e.g., 0.1 M CuSO₄ or 0.1 M NiSO₄.
5. Recharge the water-washed column by loading at least 0.5 column volume of 0.1 M metal ion/salt solution.
6. Wash with 5 column volumes of distilled water, and 5 column volumes of binding buffer (to adjust pH) before storing column in 20% ethanol.

It is important to wash with binding buffer as the last step to obtain the correct pH before storage.

Washing with buffer before applying the metal ion solution may cause unwanted precipitation.

Cleaning-in-place

When an increase in back pressure is seen, the chromatography medium should be cleaned. Before cleaning, strip off metal ions using the recommended procedure described above. The stripped medium can be cleaned by the following methods:

To remove ionically bound protein:

1. Wash with several column volumes of 1.5 to 2.0 M NaCl.
2. Immediately wash with approximately 3 to 10 column volumes of distilled water.

To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins:

1. Wash with several column volumes of 1.5 to 2.0 M NaCl.
2. Immediately wash with approximately 3 to 10 column volumes of distilled water.

To remove precipitated proteins, hydrophobically bound proteins, and lipoproteins:

1. Wash the column with 1 M NaOH, contact time usually 1 to 2 h (12 h or more for endotoxin removal).
2. Immediately wash with approximately 10 column volumes of binding buffer, followed by 5 to 10 column volumes of distilled water.

To remove hydrophobically bound proteins, lipoproteins, and lipids:

1. Wash with 5 to 10 column volumes of 30% isopropanol for about 15 to 20 min.
2. Immediately wash with approximately 10 column volumes of distilled water.
   
   2a. Alternatively, wash with 2 column volumes of detergent in a basic or acidic solution. Use, for example, 0.1 to 0.5% nonionic detergent in 0.1 M acetic acid, contact time 1 to 2 h. After treatment, always remove residual detergent by washing with at least 5 column volumes of 70% ethanol. Then wash with approximately 10 column volumes of distilled water.

Reversed flow may improve the efficiency of the cleaning-in-place procedure. After cleaning, store column in 20% ethanol (wash with 5 column volumes) or recharge with metal ions prior to storing in ethanol.