Complete analysis of tag protein purification fillers: an in-depth guide from principles to applications

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September 11, 2025

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1. Metal chelating filler

Immobilized Metal Affinity Chromatography (IMAC) is a technology widely used in the purification of proteins, especially recombinant proteins with histidine tags. The core principle is to use the affinity between amino acid residues on the surface of proteins (mainly histidine) and transition metal ions (usually nickel ions, Ni ² ⁺) immobilized on the chromatography medium for selective separation. The following is a brief introduction to three common IMAC chromatography media: Ni-IDA, Ni-NTA, and Ni-TED.

Peculiarity	Ni-IDA Sepharose FF	Ni-NTA Sepharose FF	Ni-TED Sepharose FF
Structure and Chelation	IDA (Iminodiacetic acid) is a tridentate chelating agent that binds to nickel ions through three sites (one nitrogen atom and two carboxyl oxygen atoms). This allows three remaining coordination sites on the nickel ion to bind to the histidine tag of the target protein	NTA (Nitrilotriacetic acid) is a tetradentate chelating agent that uses four sites (one nitrogen atom and three carboxyl oxygen atoms) to immobilize nickel ions. This leaves two usable binding sites for the histidine tag of the target protein	TED (Tris (carboxymethyl) ethylenediamine) is a pentadentate chelating agent that firmly chelates nickel ions through five sites, leaving only one coordination site to bind to the target protein
Affinity/Binding Loading	tall	tall	normal
Specificity	Lower. Ni ions expose more coordination sites, and the possibility of non-specific adsorption increases accordingly	High. Nickel ions are more tightly encapsulated by NTA, reducing non-specific binding	High. Minimize non-specific adsorption and obtain high-purity protein
Chelating agent tolerance	Intolerance	≤ 1 mM EDTA	≥ 10 mM EDTA
Ion leakage risk	Higher	Low	Extremely low
Applications	Suitable for conventional purification with low requirement of purity or high expression of target protein	The "gold standard" for His tag protein purification, suitable for most scenarios requiring high-purity proteins, is the most widely used IMAC medium	Suitable for purification tasks where samples contain interfering substances such as EDTA or DTT (such as animal cell expression), or where protein purity is extremely required

Ni-IDA Sepharose FF Series Use Flow Chart

Ni-NTA Sepharose FF Series Use Flow Chart

Ni-TED Sepharose FF Series Use Flow Chart

2. His tag protein purification filler parameters:

Category	Ni-IDA Sepharose FF	Ni-NTA Sepharose FF	Ni-TED Sepharose FF
Matrix	6% cross-linked agarose
Ligand	Iminodiacetic acid, ~ 30 μmol Ni2 +/mL	Nitrilotriacetic acid, ~ 17 μmol Ni2 +/mL	Tricarboxymethylethylenediamine, ~ 60 μmol Ni2 +/mL
Particle size range a	45-165 μm
Average particle size	~ 90 μm
Dynamic binding loadingb	≥ 40 mg/mL		≥ 20 mg/mL
Recommended working flow rate	60 ~ 300 cm/h
Use pH	4 ~ 8.5 (recommended working pH), 3 ~ 12 (long-term stability); 2 ~ 14 (short-term stable)
Chemical stability	Stable in the following solutions: commonly used aqueous phase buffer, 1 mol/L sodium hydroxide, 8 mol/L urea, 6 mol/L guanidine hydrochloride, 70% ethanol, etc.

Note:

A: More than 90% by volume of the microspheres are in this particle size range;

B: DBC 10% test conditions were: 10% flow-through, E. coli expression of histidine-tagged recombinant protein, 6min residence time.

3. Application data:

Figure 4

Lane M： Marker

Lane 1 ：Start material

Lane 2: Flowthrough 1

Lane 3: Flowthrough 2

Lane 4: Elution (20 mM Imidazole)

Lane 5: Elution (50 mM Imidazole)

Lane 6: Elution (100 mM Imidazole)

Lane 7: Elution (200 mM Imidazole)

Lane 8: Elution (300 mM Imidazole)

Lane 9: Elution (500 mM Imidazole)

Packing: Ni-IDA Sepharose FF (Gravity Preloaded Column) (Cat: abs91007-G), 1 mL

Sample: his6-tagged recombinant Streptococcus Protein G produced in E. coli

Binding Buffer : 20 mM PB, 150 mM NaCl, pH 7.4

Elution Buffer : 20 mM PB, 150 mM NaCl, 20-500 mM Imidazole, pH 7.4

Figure 5

Packing: Ni-NTA Sepharose FF (Medium Pressure Preloaded Column) (Cat: abs91008-M), 1 mL

Sample: his6-tagged recombinant Streptococcus Protein G produced in E. coli

Binding Buffer : 20 mM PB, 150 mM NaCl, pH 7.4

Elution Buffer : 20 mM PB, 150 mM NaCl, 20-500 mM Imidazole, pH 7.4

Affinity Purification of Other Tag Proteins

Peculiarity	GST Sepharose FF	MBP Sepharose FF	Biotin Sepharose FF	Strep II Sepharose FF
Filler ligand	Glutathione	Dextrin	Streptavidin	Streptavidin mutant
Label of action	GST (Glutathione S-transferase), ~ 26 kDa	MBP (Maltose-Binding Protein), ~ 42 kDa Protein of	Biotin labels (site-directed ligation by biotin ligase (BirA) to specific sequences of the target protein (e.g. AviTag), can also be non-specifically ligated by chemical methods	Strep II, a short peptide of 8 amino acids (WSHPQFEK)
Purification principle	GST tag can specifically and reversibly bind to glutathione immobilized on medium	The MBP tag is able to specifically recognize and bind dextrin on the medium	There is one of the strongest non-covalent interactions in nature between biotin and streptavidin	The Strep II tag enables high-affinity, high-specificity reversible binding to the biotin-binding pocket of the streptavidin mutant
Elution Method	Competitive washing using Reduced Glutathione	Competitive elution using a high concentration of Maltose solution	Denaturing elution	Competitive elution using D-biotin solution

GST Sepharose FF Application:

Figure 6

Lane M： Marker

Lane 1： Start material

Lane 2: Flowthrough

Lane 3: Elution

Packing: GST Sepharose FF (Gravity Pre-Packed Column) (Cat: abs91010-G), 1 mL

Sample: GST-SUMO produced in E. coli

Binding Buffer : 20 mM PB, 150 mM NaCl, pH 7.4

Elution Buffer : 20 mM PB, 150 mM NaCl, 10 mM glutathione, pH 7.4

MBP Sepharose FF Application:

Figure 7

Lane M： Marker

Lane 1： Start material

Lane 2: Flowthrough

Lane 3: Elution

Packing: MBP Sepharose FF (Gravity Pre-Packed Column) (Cat: abs91011-G), 1 mL

Sample: MBP produced in E. coli

Binding Buffer : 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.4

Elution Buffer : 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 mM D-maltose, pH7.4

Figure 8

Lane M： Marker

Lane 1： Start material

Lane 2: Flowthrough

Lane 3: Elution

Packing: Strep II Sepharose FF (Gravity Preloaded Column) (Cat: abs91013-G), 1 mL

Sample: Strep II-tagged EGF produced in E. coli

Binding Buffer : 20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, pH 8.0

Elution Buffer : 20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, 10 mM D-Biotin, pH 8.0

4. Absin label protein purification filler product information

Item Number	Trade Name
abs91008-G	Ni-NTA Sepharose FF (Gravity Pre-Packed Column)
abs91008-M	Ni-NTA Sepharose FF (Medium Pressure Pre-loaded Column)
abs91008-F	Ni-NTA Sepharose FF
abs91007-G	Ni-IDA Sepharose FF (Gravity Preloaded Column)
abs91007-M	Ni-IDA Sepharose FF (Medium Pressure Pre-loaded Column)
abs91007-F	Ni-IDA Sepharose FF
abs91009-F	Ni-TED Sepharose FF
abs91009-G	Ni-TED Sepharose FF (Gravity Preloaded Column)
abs91009-M	Ni-TED Sepharose FF (Medium Pressure Pre-loaded Column)
abs91010-F	GST Sepharose FF
abs91010-G	GST Sepharose FF (Gravity Pre-Packed Column)
abs91010-M	GST Sepharose FF (Medium Pressure Pre-loaded Column)
abs91011-F	MBP Sepharose FF
abs91011-G	MBP Sepharose FF (Gravity Pre-Packed Column)
abs91011-M	MBP Sepharose FF (Medium Pressure Pre-loaded Column)
abs91012-F	Biotin Sepharose FF
abs91012-G	Biotin Sepharose FF (Gravity Pre-Packed Column)
abs91012-M	Biotin Sepharose FF (Medium Pressure Pre-loaded Column)
abs91013-F	Strep II Sepharose FF
abs91013-G	Strep II Sepharose FF (Gravity Preloaded Column)
abs91013-M	Strep II Sepharose FF (Medium Pressure Pre-loaded Column)

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