Rprotein a sepharose 4 fast flow

Manufactured by GE Healthcare

Sourced in United Kingdom

RProtein A Sepharose 4 Fast Flow is a pre-packed affinity chromatography medium designed for the purification of immunoglobulins and Fc-containing proteins. It is based on highly cross-linked agarose beads with covalently coupled recombinant Protein A ligand, providing a high dynamic binding capacity and fast flow properties.

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Lab products found in correlation

Resource s column, by GE Healthcare (1 mentions) Nhs ss peg4 biotin, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Sepharose 4 Fast Flow (37 citations) RProtein A Sepharose (24 citations) RProtein A Sepharose Fast Flow (12 citations) RProtein A (5 citations)

3 protocols using rprotein a sepharose 4 fast flow

Antibody Selection against Ci-VSD Mutants

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WT and R217E mutant of Ci-VSD-1-260 constructs were biotinylated through amine
coupling with NHS-SS-PEG4-Biotin (Thermo Scientific). Phage display selections were
performed using a synthetic antibody library built on the 4D5 Fab scaffold as described
previously (Rizk et al., 2011 (link)). Target protein
concentrations were serially adjusted using 100 nM in the first round and 10 nM and 5 nM
in subsequent rounds to ensure proper stringency. The conformation specific binders were
selected using a competitive selection strategy where 1 μM nonbiotinylated
competitor (such as Ci-VSD WT) was added to the phage library prior to the incubation with
actual selection target (such as Ci-VSD R217E). A single point competitive ELISA step used
to identify positive clones. Fabs were expressed in E. coli strain 55244 in phosphate
depleted media as described previously (Rizk et al.,
2011). Fabs were purified using automated two step protocol employing a custom
build AKTA purification system with 5ml rProteinA Sepharose 4 Fast Flow and 1ml Resource S
column (GE Healthcare) columns. Fractions were verified by SDS-PAGE, pooled and dialyzed
against PBS.

Li Q., Wanderling S., Paduch M., Medovoy D., Singharoy A., McGreevy R., Villalba-Galea C., Hulse R.E., Roux B., Schulten K., Kossiakoff A, & Perozo E. (2014). Structural Mechanism of Voltage-Dependent Gating in an Isolated Voltage-Sensing Domain. Nature structural & molecular biology, 21(3), 244-252.

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Small-Scale Antibody Purification from Leaves

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Small-scale antibody purification was performed essentially as described previously [17 (link)]. Briefly, frozen leaf material was first crushed in a ball mill as above and then extracted with 2 μL buffer (45 mM tris/HCl (pH 7.4), 1.5 M NaCl, 40 mM ascorbic acid, 1 mM EDTA) per mg of leaf material for 15 min at 0°C. After centrifugation (5 min, 14000g, 4°C), the supernatant was incubated with rProtein A Sepharose 4 Fast Flow (GE Healthcare, Little Chalfont, UK) for 90 min under constant agitation at 4°C. The beads were then collected by centrifugation and washed with phosphate-buffered saline (PBS) prior to elution of bound antibody with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer.

Niemer M., Mehofer U., Torres Acosta J.A., Verdianz M., Henkel T., Loos A., Strasser R., Maresch D., Rademacher T., Steinkellner H, & Mach L. (2014). The human anti-HIV antibodies 2F5, 2G12, and PG9 differ in their susceptibility to proteolytic degradation: Down-regulation of endogenous serine and cysteine proteinase activities could improve antibody production in plant-based expression platforms. Biotechnology Journal, 9(4), 493-500.

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Antibody Extraction and Purification

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Antibody extraction and purification was performed
as described previously, with minor modifications.^{41 (link),43 (link)} Briefly, infiltrated leaf material was homogenized under liquid
nitrogen and extracted with 0.1 M Tris/HCl (pH 7.0) containing 0.5
M NaCl, 40 mM ascorbic acid and 1 mM EDTA (2 mL per gram leaf wet
weight). The extract was clarified by a series of centrifugation and
filtration steps. Antibodies were then purified by affinity chromatography
on a column packed with 1 mL rProtein A Sepharose 4 Fast Flow (GE
Healthcare, UK), using 0.1 M glycine/HCl (pH 3.0) for elution. Protein-containing
eluate fractions were immediately neutralized by addition of 0.1 M
Tris/HCl (pH 8.0), dialyzed against PBS containing 0.02% (v/v) NaN₃ and then concentrated by ultrafiltration using Amicon YM30
centrifugal filter units (Merck, Germany).

Trattnig N., Mayrhofer P., Kunert R., Mach L., Pantophlet R, & Kosma P. (2018). Comparative Antigenicity of Thiourea and Adipic Amide Linked Neoglycoconjugates Containing Modified Oligomannose Epitopes for the Carbohydrate-Specific anti-HIV Antibody 2G12. Bioconjugate Chemistry, 30(1), 70-82.

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