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Streptavidin sepharose

Manufactured by GE Healthcare
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Streptavidin Sepharose is a versatile affinity resin used for the purification of biotinylated molecules. It consists of streptavidin, a protein with a high affinity for biotin, covalently coupled to Sepharose beads. The core function of Streptavidin Sepharose is to capture and isolate biotinylated proteins, nucleic acids, and other biomolecules from complex samples.

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Lab products found in correlation

Xk column, by GE Healthcare (9 mentions) Complete protease inhibitor, by Roche (4 mentions) Biotinylation kit, by Avidity (4 mentions) Pyromark gold q96 reagent, by Qiagen (4 mentions) Hotstartaq dna polymerase, by Qiagen (3 mentions) Hotstar taq pcr buffer, by Qiagen (3 mentions) Pyromark buffers, by Qiagen (3 mentions) Protease inhibitor cocktail, by Merck Group (3 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Sulfo nhs ss biotin, by Thermo Fisher Scientific (3 mentions) Ez dna methylation kit, by Zymo Research (2 mentions) Psq 96ma, by Qiagen (2 mentions) Protein a sepharose, by GE Healthcare (2 mentions) Ltq orbitrap, by Thermo Fisher Scientific (2 mentions) Dtssp, by Thermo Fisher Scientific (2 mentions) 3d7 sequence database version 3, by Matrix Science (2 mentions) Mascot software, by Matrix Science (2 mentions) Bira biotin protein ligase, by Avidity (2 mentions) Odyssey scanner, by LI COR (2 mentions) Phosstop, by Roche (2 mentions)

Spelling variants of this product

Streptavidin Sepharose beads (116 citations) Streptavidin-coated Sepharose beads (23 citations) Streptavidin Sepharose HP beads (12 citations) Streptavidin-coated Sepharose high-performance beads (6 citations) Streptavidin-conjugated sepharose beads (5 citations) Streptavidin magnetic sepharose beads (4 citations) Streptavidin magnetic Sepharose (2 citations) One gram streptavidin sepharose (2 citations) Streptavidin Sepharose High Performance medium (2 citations) Streptavidin-sepharose matrix (1 citations)

75 protocols using streptavidin sepharose

1

Affinity Chromatography of Fc-RN Bispecific Antibodies

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Example 5

Fc-Rn Chromatography

Coupling to Streptavidin Sepharose:

One gram Streptavidin Sepharose® (GE Healthcare) was added to the biotinylated and dialyzed receptor and incubated for two hours with shaking. The receptor derivatized sepharose was filled in a 1 ml XK column (GE Healthcare).

Chromatography Using the FcRn Affinity Column:

Conditions:

column dimensions: 50 mm×5 mm

bed height: 5 cm

loading: 50 μg sample

equilibration buffer: 20 mM MES, with 150 mM NaCl, adjusted to pH 5.5

elution buffer: 20 mM Tris/HCl, with 150 mM NaCl, adjusted to pH 8.8

elution: 7.5 CV equilibration buffer, in 30 CV to 100% elution buffer, 10 CV elution buffer

Hu FcRn Affinity Column Chromatography

In the following table retention times of <VEGF-ANG-2> bispecific antibodies on affinity columns comprising human FcRn are given. Data were obtained using the conditions above. In the following Table retention times of <VEGF-ANG-2> bispecific antibodies on human FcRn are given.

TABLE 12
Results: retention times of <VEGF-ANG-2> bispecific antibodies
antibodyretention time [min]
VEGFAng2-0015 (without AAA mutation)78.5
VEGFAng2-0201 (without AAA mutation)78.9
VEGFAng2-0012 (with AAA mutation)2.7 (Void-peak)
VEGFAng2-0016 (with AAA mutation)2.7 (Void-peak)

US10683345B2. Bispecific anti-VEGF/anti-ANG-2 antibodies and their use in the treatment of ocular vascular diseases (2020-06-16). Roche Glycart AG [CH]. Inventors: Harald Duerr [DE], Frank Herting [DE], Christian Klein [CH], Joerg Thomas Regula [DE], Matthias Rueth [DE], Kay-Gunnar Stubenrauch [DE].
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2

FcRn Affinity Chromatography of Anti-VEGF/ANG2 Antibodies

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Example 5

FcRn Chromatography

Coupling to Streptavidin sepharose:

One gram Streptavidin sepharose (GE Healthcare) was added to the biotinylated and dialyzed receptor and incubated for two hours with shaking. The receptor derivatized sepharose was filled in a 1 mL XK column (GE Healthcare).

Chromatography Using the FcRn Affinity Column:

  • Conditions:
  • column dimensions: 50 mm×5 mm
  • bed height: 5 cm
  • loading: 50 μg sample
  • equilibration buffer: 20 mM MES, with 150 mM NaCl, adjusted to pH 5.5
  • elution buffer: 20 mM Tris/HCl, with 150 mM NaCl, adjusted to pH 8.8
  • elution: 7.5 CV equilibration buffer, in 30 CV to 100% elution buffer, 10 CV elution buffer
    Human FcRn Affinity Column Chromatography

In the following Table of retention times of anti-VEGF/ANG2 antibodies on affinity columns comprising human FcRn are given. Data were obtained using the conditions above.

TABLE
Results: retention times of anti-VEGF/ANG2 antibodies
antibodyretention time [min]
VEGF/ANG2-0015 (without78.5
IHH-AAA mutation)
VEGF/ANG2-0201 (without78.9
IHH-AAA mutation)
VEGF/ANG2-0012 (with IHH-2.7 (void-peak)
AAA mutation)
VEGF/ANG2-0016 (with IHH-2.7 (void-peak)
AAA mutation)

US11555067B2. Fc-region variants with improved protein A-binding (2023-01-17). Hoffmann-La Roche Inc. [US]. Inventors: Tilman Schlothauer [DE].
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3

Affinity Purification of FcRn Protein

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Example 2

FcRn Chromatography

Coupling to Streptavidin Sepharose:

One gram Streptavidin Sepharose (GE Healthcare) is added to the biotinylated and dialyzed receptor and incubated for two hours with shaking. The receptor derivatized Sepharose is filled in a 1 mL XK column (GE Healthcare).

Chromatography Using the FcRn Affinity Column:

    • Conditions:
    • column dimensions: 50 mm×5 mm
    • bed height: 5 cm
    • loading: 50 μg sample
    • equilibration buffer: 20 mM MES, with 150 mM NaCl, adjusted to pH 5.5
    • elution buffer: 20 mM Tris/HC1, with 150 mM NaCl, adjusted to pH 8.8
    • elution: 7.5 CV equilibration buffer, in 30 CV to 100% elution buffer, 10 CV elution buffer

US10899846B2. Fc-region variants with modified FcRn- and protein A-binding properties (2021-01-26). Hoffmann-La Roche Inc. [US]. Inventors: Tilman Schlothauer [DE].
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4

Affinity Purification of Rat zDHHC5 Interactors

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An array of 25 amino acid peptides overlapping by 15 residues each, representing the C tail of rat zDHHC5 was purchased from Alta Bioscience. Each peptide had an N terminal biotin for affinity purification. Rat ventricular lysates were precleared by incubation with streptavidin Sepharose (GE Life Sciences) for 1–2 h at 4 °C, incubated with 1 μg peptide (~0.2 μM final concentration) overnight at 4 °C with end-over-end mixing, and the peptides purified the next day with streptavidin Sepharose. After extensive washing peptide–protein complexes were eluted with SDS PAGE loading buffer.
Plain F., Howie J., Kennedy J., Brown E., Shattock M.J., Fraser N.J, & Fuller W. (2020). Control of protein palmitoylation by regulating substrate recruitment to a zDHHC-protein acyltransferase. Communications Biology, 3, 411.
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5

Facile FcγRIIIaV158 Affinity Column

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Example 7

Preparation of FcgammaRIIIaV158 Affinity Column

An affinity column with FcgammaRIIIaV158 was prepared by in vitro biotinylation of the Avi-tag and subsequent coupling to Streptavidin Sepharose®. This can be done with the intact fusion polypeptide as well as with the receptor after having cleaved off the Fc-region. It is a very quick and efficient method for preparing an affinity column for analytical and preparative purposes.

Biotinylation of Receptor

A soluble extracellular domain of FcgammaRIIIaV158 with Avi Tag expressed in HEK293 cells was biotinylated after purification according to the following protocol: between 1.2 and 12 mg FcgammaRIIIaV158 or between 2.4 and 24 mg FcgammaRIIIaV158 Fc-region fusion polypeptide tagged in 2 mM MOPS, 125 mM NaCl pH 7.2, 0.02% Tween™, and 1 tablet Complete protease inhibitor (Roche) in 3 ml PBS were biotinylated using the biotinylation kit from Avidity according to the manufacturer instructions. Biotinylation reaction was done at room temperature overnight. The modified polypeptide was dialyzed against 20 mM sodium phosphate buffer, 150 mM NaCl pH 7.5 at 4° C. overnight to remove excess biotin.

Coupling to Streptavidin Sepharose®

1 g Streptavidin Sepharose® (GE Healthcare) was added to the biotinylated and dialyzed receptor, incubated for 2 hours while shaking and finally filled in a 1 ml XK column (GE Healthcare).

US10011644B2. Method for producing soluble FcR as Fc-fusion with inert immunoglobulin Fc-region and uses thereof (2018-07-03). Hoffmann-La Roche Inc. [US]. Inventors: Petra Rueger [DE], Tilman Schlothauer [DE], Stefan Seeber [DE].
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6

Preparation of FcgammaRIIIaV158 Affinity Column

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Example 7

Preparation of FcgammaRIIIaV158 Affinity Column

An affinity column with FcgammaRIIIaV158 was prepared by in vitro biotinylation of the Avi-tag and subsequent coupling to Streptavidin Sepharose®. This can be done with the intact fusion polypeptide as well as with the receptor after having cleaved off the Fc-region. It is a very quick and efficient method for preparing an affinity column for analytical and preparative purposes.

Biotinylation of Receptor

A soluble extracellular domain of FcgammaRIIIaV158 with Avi Tag expressed in HEK293 cells was biotinylated after purification according to the following protocol: between 1.2 and 12 mg FcgammaRIIIaV158 or between 2.4 and 24 mg FcgammaRIIIaV158 Fc-region fusion polypeptide tagged in 2 mM MOPS, 125 mM NaCl pH 7.2, 0.02% Tween™, and 1 tablet Complete protease inhibitor (Roche) in 3 ml PBS were biotinylated using the biotinylation kit from Avidity according to the manufacturer instructions. Biotinylation reaction was done at room temperature overnight. The modified polypeptide was dialyzed against 20 mM sodium phosphate buffer, 150 mM NaCl pH 7.5 at 4° C. overnight to remove excess biotin.

Coupling to Streptavidin Sepharose®

1 g Streptavidin Sepharose® (GE Healthcare) was added to the biotinylated and dialyzed receptor, incubated for 2 hours while shaking and finally filled in a 1 ml XK column (GE Healthcare).

US10570188B2. Method for producing monomeric and multimeric molecules and uses thereof (2020-02-25). Hoffmann-La Roche Inc. [US]. Inventors: Johannes Auer [DE], Martin Bader [DE], Stefan Dengl [DE], Stefan Lorenz [DE], Stefan Seeber [DE].
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7

Preparation of FcgammaRIIIaV158 Affinity Column

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Example 7

Preparation of FcgammaRIIIaV158 Affinity Column

An affinity column with FcgammaRIIIaV158 was prepared by in vitro biotinylation of the Avi-tag and subsequent coupling to Streptavidin Sepharose®. This can be done with the intact fusion polypeptide as well as with the receptor after having cleaved off the Fc-region. It is a very quick and efficient method for preparing an affinity column for analytical and preparative purposes.

Biotinvlation of Receptor

A soluble extracellular domain of FcgammaRIIIaV158 with Avi Tag expressed in HEK293 cells was biotinylated after purification according to the following protocol: between 1.2 and 12 mg FcgammaRIIIaV158 or between 2.4 and 24 mg FcgammaRIIIaV158 Fc-region fusion polypeptide tagged in 2 mM MOPS, 125 mM NaCl pH 7.2, 0.02% Tween™, and 1 tablet Complete protease inhibitor (Roche) in 3 ml PBS were biotinylated using the biotinylation kit from Avidity according to the manufacturer instructions. Biotinylation reaction was done at room temperature overnight. The modified polypeptide was dialyzed against 20 mM sodium phosphate buffer, 150 mM NaCl pH 7.5 at 4° C. overnight to remove excess biotin.

Coupling to Streptavidin Sepharose®

1 g Streptavidin Sepharose® (GE Healthcare) was added to the biotinylated and dialyzed receptor, incubated for 2 hours while shaking and finally filled in a 1 ml XK column (GE Healthcare).

US11254728B2. Method for producing monomeric and multimeric molecules and uses thereof (2022-02-22). Hoffmann-La Roche Inc. [US]. Inventors: Johannes Auer [DE], Martin Bader [DE], Stefan Dengl [DE], Stefan Lorenz [DE], Stefan Seeber [DE].
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8

Affinity Column Preparation with FcγRIIIaV158

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Example 7

Preparation of FcgammaRIIIaV158 Affinity Column

An affinity column with FcgammaRIIIaV158 was prepared by in vitro biotinylation of the Avi-tag and subsequent coupling to Streptavidin Sepharose®. This can be done with the intact fusion polypeptide as well as with the receptor after having cleaved off the Fc-region. It is a very quick and efficient method for preparing an affinity column for analytical and preparative purposes.

Biotinylation of Receptor

A soluble extracellular domain of FcgammaRIIIaV158 with Avi Tag expressed in HEK293 cells was biotinylated after purification according to the following protocol: between 1.2 and 12 mg FcgammaRIIIaV158 or between 2.4 and 24 mg FcgammaRIIIaV158 Fc-region fusion polypeptide tagged in 2 mM MOPS, 125 mM NaCl pH 7.2, 0.02% Tween™, and 1 tablet Complete protease inhibitor (Roche) in 3 ml PBS were biotinylated using the biotinylation kit from Avidity according to the manufacturer instructions. Biotinylation reaction was done at room temperature overnight. The modified polypeptide was dialyzed against 20 mM sodium phosphate buffer, 150 mM NaCl pH 7.5 at 4° C. overnight to remove excess biotin.

Coupling to Streptavidin Sepharose®

1 g Streptavidin Sepharose® (GE Healthcare) was added to the biotinylated and dialyzed receptor, incubated for 2 hours while shaking and finally filled in a 1 ml XK column (GE Healthcare).

US10093714B1. Method for producing soluble FcR as Fc-fusion with inert immunoglobulin Fc-region and uses thereof (2018-10-09). Hoffmann-La Roche Inc. [US]. Inventors: Petra Rueger [DE], Tilman Schlothauer [DE], Stefan Seeber [DE].
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9

Affinity Purification of SFB-Tagged RIP1

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HEK293T cells were transfected with plasmid encoding SFB-RIP1. After 24 hours, cell lysates were harvested by IP lysis buffer first incubated with streptavidin Sepharose (GE Healthcare) for 4 hours at 4°C, and after several washes, streptavidin Sepharose–bound proteins were eluted with biotin (1 mg/ml). Then, the eluent was incubated with S-protein agarose beads (Novagen) overnight at 4°C. After being washed for three times, immobilized proteins were eluted in SDS sample loading buffer and separated by SDS-PAGE. The protein mixtures were analyzed by nano–liquid chromatography–tandem mass spectrometry analysis for protein identification at Phoenix National Proteomics Core service as described (52 (link)).
Gao C., Deng J., Zhang H., Li X., Gu S., Zheng M., Tang M., Zhu Y., Lin X., Jin J., Zhang L., Huang J., Zou J., Xia Z.P., Xu P.L., Shen L., Zhao B, & Feng X.H. (2021). HSPA13 facilitates NF-κB–mediated transcription and attenuates cell death responses in TNFα signaling. Science Advances, 7(41), eabh1756.
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10

Cell Surface Biotinylation and Protein Quantification

Cited 1 time
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For non-quantitative analyses cell surface biotinylation and subsequent streptavidin-precipitation was performed as described previously [34] (link). After cell lysis the samples were divided into equal parts for immunoprecipitation with either Flag-Agarose Beads (M2, Sigma) to detect DLL1 irrespective of its cellular localization and Streptavidin-Sepharose (GE) to detect the surface biotinylated fraction. For quantitative analyses 0,5×106 mouse fibroblast cells were plated on 6-cm dishes and biotinylated as described [34] (link). After cell lysis a 50 µl aliquot was taken and mixed with 2 x sample buffer. The remaining lysate was used for immunoprecipitation (IP) with NeutrAvidin Agarose Resin (Thermo Scientific, 29200) over night at 4°C. The resin was washed three times in Ripa lysis buffer (50 mM Tris/HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1% TritonX100, 0.25% DOC and 0.1% SDS supplemented with Protease Inhibitor Cocktail tablets (Roche, 04693159)) and resuspended in 2 x sample buffer. The supernatant of the IP was subjected to a second round of IP to confirm that DLL1 was quantitatively precipitated in the first IP. Protein from whole cell lysates and immunoprecipitated proteins were subjected to SDS-PAGE and analyzed by Western blotting using the Fuji LAS-4000 Western blot detection system. Protein bands were quantitated using ImageJ [36] (link).
Müller J., Rana N.A., Serth K., Kakuda S., Haltiwanger R.S, & Gossler A. (2014). O-fucosylation of the Notch Ligand mDLL1 by POFUT1 Is Dispensable for Ligand Function. PLoS ONE, 9(2), e88571.
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