Ni-NTA Agarose for His-tagged proteins
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Product information "Ni-NTA Agarose for His-tagged proteins"
Ni-NTA agarose was developed for affinity purification of proteins bearing a polyhistidine label. This affinity chromatography matrix is based on BioWorks workbeads consisting of 7.5% cross-linked agarose. The material is highly porous to allow optimal protein interaction. Cross-linked agarose is also physically very stable and suitable for low pressure purification processes with flow rates up to 6mL/min (optimal 0.5 - 2mL/min).
Our Ni-NTA agarose has a very homogeneous size with a mean particle diameter of 40μm, resulting in a high degree of reproducibility between individual purification runs.
Ni-NTA agarose consists of cross-linked agarose to which tetradendritic nitrilotriacetic acid (NTA) loaded with Ni2+ ions is covalently bound. The binding capacity varies with different proteins based on their specific properties, but it is at least 50mg target protein per mL agarose gel. The resin is ideally suited for batch, spin and column applications of any scale and offers a high degree of reproducibility between individual purification approaches with a very homogeneous size distribution around a mean particle diameter of 40µm.
The Ni-NTA agarose is supplied as a buffered suspension in 50% ethanol. For example, 1x S5377.0010 corresponds to a suspension of 10mL resin and 10mL water with 20% EtOH. Total supplied volumes is 20mL.
Specifications
cross-linked Agarose
particle size: 32-60µm
binding capacity: 50mg/mL resin
max pressure: 3 bar (43 psi)
pH-stability: 2-14 (short term)
pH-stability: 3–12 (long term)
flow rates: 0.5-2.0mL/min (normally) up to 6.0mL/min possible
stable against: 100% MeOH, 100% EtOH, 8M Urea, 6M Guanidinium hydrochloride, 30% (v/v) Acetonitrile
Applikation / Application:
for purification of recombinant proteins for purification of His-tagged proteinsQuelle / Source:
syntheticKlassifizierungen / Classification
Documents:
Safety Data SheetProtocols
Certificate
Manuals
Product description
General Data 1
Die Reinigung von His-getaggten Proteinen erfolgt typischerweise über Immobilized Metal Affinity Chromatography (IMAC), bei der Metallionen wie Ni²⁺ (Nickel) oder Co²⁺ (Cobalt) an eine Matrix (z. B. NTA oder IDA) gebunden sind. Die Unterschiede zwischen Ni-Agarose und Co-Agarose bei der Reinigung ergeben sich hauptsächlich aus:
1. Bindungsspezifität
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Ni²⁺ (Nickel-Agarose):
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Geringere Spezifität: Nickel bindet auch Proteine mit nur wenigen Histidinen oder anderen Seitenketten (z. B. Cystein, Tryptophan).
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Mehr unspezifische Bindung, daher höhere Ausbeute, aber auch mehr Kontamination.
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Co²⁺ (Cobalt-Agarose):
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Höhere Spezifität: Bindet bevorzugt an vollständige His-Tags (z. B. 6xHis).
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Weniger unspezifische Bindung, daher reineres Eluat, aber tendenziell niedrigere Ausbeute.
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2. Ausbeute vs. Reinheit
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Ni-Agarose: Höhere Ausbeute, geringere Reinheit.
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Co-Agarose: Geringere Ausbeute, höhere Reinheit.
3. Elutionsverhalten
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Beide Systeme verwenden imidazolhaltige Puffer zur Elution, aber:
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Co²⁺-Säulen benötigen oft niedrigere Imidazolkonzentrationen zur Elution des Zielproteins.
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Dies kann empfindlicheren Proteinen zugutekommen.
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4. Empfindlichkeit gegenüber Imidazol im Waschpuffer
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Co²⁺-Säulen reagieren empfindlicher auf Imidazol in der Waschphase und verlieren bei zu hoher Konzentration schneller das Zielprotein.
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Ni²⁺-Säulen sind toleranter und ermöglichen aggressiveres Waschen zur Entfernung von Kontaminanten.
Source: NCBI PubMed
Quelle/Source:NCBI PubMed >
Pavel Bashtrykov, Gytis Jankevicius, Renata Z. Jurkowska, Sergey Ragozin, Albert Jeltsch
J Biol Chem. 2014 Feb 14; 289(7): 4106–4115. Published online 2013 Dec 24. doi: 10.1074/jbc.M113.528893
PMCID: PMC3924276
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