Biobeads

Manufactured by GE Healthcare

BioBeads are a versatile and advanced protein purification system developed by GE Healthcare. The core function of BioBeads is to efficiently capture and separate target proteins from complex mixtures, enabling effective purification for a wide range of applications.

Automatically generated - may contain errors

Lab products found in correlation

Bio beads, by Bio-Rad (5 mentions) Amphipol a8 35, by Anatrace (1 mentions) Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions) Asolectin lipids, by Merck Group (1 mentions) Biobeads, by Merck Group (1 mentions) Superose 6 increase column, by GE Healthcare (1 mentions) Superose 6 column, by GE Healthcare (1 mentions) Cholate, by Merck Group (1 mentions) Superdex 200 increase column, by GE Healthcare (1 mentions) Superdex 200 increase 10 300, by GE Healthcare (1 mentions) 1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions) 1 2 dioleoyl sn glycero 3 phospho l serine, by Avanti Polar Lipids (1 mentions) Amicon centrifugal concentrator, by Merck Group (1 mentions) Superose 6 increase 10 300 gl column, by GE Healthcare (1 mentions)

6 protocols using biobeads

Amphipol-mediated VanSA Protein Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Purified, concentrated VanS_A protein (approx. 150 uM) containing approximately 120 mM LDAO was diluted to 22 uM (1 mg/mL) with 50 mM Tris pH 7.4, 50 mM KCl. An aliquot of a 10% w/v solution of amphipol A8-35 (Anatrace) was added to give a 1:1 mass ratio of protein to amphipol [34 ], and the solution was incubated on ice for 30 minutes. A quantity of washed BioBeads (BioRad) was added equivalent to 20x the estimated mass of LDAO present, and the solution was mixed at 4° for 2 hours. Pilot ultracentrifugation experiments were used to determine that this treatment gave 100% recovery of the VanS_A protein in the supernatant. Once this was confirmed, subsequent experiments omitted the ultracentrifugation step. After detergent removal with the BioBeads, the material was injected onto a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated with 20 mM Tris-Cl pH 7.4, 150 mM NaCl, and the peak corresponding to active VanS_A was collected and used for enzymatic assays. A second, later-eluting peak was also observed, but showed substantially lower activity. The nature of this second species was not investigated further, but we did note that the second peak showed almost no dimer band on SDS-PAGE gels, suggesting it might containing VanS_A monomers. Addition of 1% C12E8 to the protein solution prior to amphipol reconstitution decreased the relative size of the second peak.

Upton E.C., Maciunas L.J, & Loll P.J. (2019). Vancomycin does not affect the enzymatic activities of purified VanSA. PLoS ONE, 14(1), e0210627.

+ Open protocol

+ Expand

5-HT3A Receptor Reconstitution in Nanodiscs

Check if the same lab product or an alternative is used in the 5 most similar protocols

All steps were performed at 4 °C. The peak fractions after size-exclusion chromatography in the C12E9 detergent (Anatrace) containing 5-HT3A receptor were pooled and concentrated to 1 mg/ml and mixed with asolectin lipids (Sigma-Aldrich) solubilized at 5 mg/ml in 5% DDM (Anatrace). After 30 minutes incubation, MSP1E3D1(-) (a gift from Stephen Sligar, Addgene plasmid #20066, expressed and purified as previously described^{98 (link)}) was added to the mixture, which was incubated for 30 additional minutes, before the addition of Bio-Beads (Sigma-Aldrich) at 10 mg/ml final concentration. The molar ratio of the receptor over the MSP and the lipids was 1:7:200. The mixture was incubated under gentle rotation overnight for detergent removal and nanodiscs reconstitution. Bio-Beads were removed by centrifugation (250g, 10 min) and the supernatant was subjected to size-exclusion chromatography in a Superose 6 Increase column (GE healthcare) equilibrated in SEC buffer (50 mM Tris-HCl, 125 mM NaCl, pH 7.5). The fractions containing the reconstituted receptor in nanodiscs were pooled, concentrated to 0.5 mg/ml, aliquoted, snap frozen in liquid nitrogen and stored at -80°C.

Uchański T., Masiulis S., Fischer B., Kalichuk V., López-Sánchez U., Zarkadas E., Weckener M., Sente A., Ward P., Wohlkönig A., Zögg T., Remaut H., Naismith J.H., Nury H., Vranken W., Aricescu A.R., Pardon E, & Steyaert J. (2021). Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM. Nature methods, 18(1), 60-68.

+ Open protocol

+ Expand

Reconstitution of AdeJ into Nanodiscs

Check if the same lab product or an alternative is used in the 5 most similar protocols

AdeJ was assembled into nanodiscs using a mixture of 20 μM AdeJ, 45 μM membrane scaffold protein (MSP) (1E3D1), and 930 μM E. coli total lipid extract. The mixture was incubated at room temperature for 15 min. To remove the detergent, 0.8 mg/mL prewashed Bio-Beads (Bio-Rad) was added. The mixture was incubated on ice for 1 h, followed by incubation at 4°C overnight. The sample was filtered using a 0.22-μm nitrocellulose filter to remove Bio-Beads and purified from empty nanodiscs using a Superose 6 column (GE Healthcare) in a solution containing 20 mM Tris-HCl (pH 7.5) and 100 mM NaCl. Fractions containing the trimeric AdeJ-nanodisc complex were collected for cryo-EM sample preparation.

Morgan C.E., Zhang Z., Bonomo R.A, & Yu E.W. (2022). An Analysis of the Novel Fluorocycline TP-6076 Bound to Both the Ribosome and Multidrug Efflux Pump AdeJ from Acinetobacter baumannii. mBio, 13(1), e03732-21.

+ Open protocol

+ Expand

Nanodisc Preparation with POPC and POPS

Check if the same lab product or an alternative is used in the 5 most similar protocols

POPC and POPS lipids were purchased from Avanti Polar Lipids in chloroform with concentrations determined by total phosphate analysis. Nanodiscs containing 100% POPC and 70% POPC/30% POPS were prepared based on a published protocol with minor modifications^{76 (link)}. Briefly, stock lipids were mixed together at room temperature, slowly dried using argon gas while incubating in a bead heat bath at 37 °C, and then put on a vacuum lyophilyzer overnight to remove all residual organic solvents. The dried lipids were reconstituted to 65 mM with 130 mM cholate (Sigma -Aldrich) in 20 mM HEPES pH 7.4, 100 mM NaCl, and 0.5 mM EDTA buffer, and mixed with MSP ΔH5 in a 30:1 lipid to protein ratio. The mixtures were mixed on a Nutator at 4 °C for 1 hour, and then 0.6 g/mL of washed bio-beads (Bio-Rad laboratory) were added and incubated at 4 °C for additional 4.5 hours. Afterwards, the nanodiscs were removed from the bio-beads by careful pipetting and purified using gel filtration chromatography on an AKTA FPLC with a GE Superdex 200 increase column (10 × 300 mm) with the mobile buffer containing 20 mM HEPES pH 7.4, 100 mM NaCl, and 0.5 mM EDTA at 0.5 mL/min flow rate. Peak fractions were pooled and analyzed by dynamic light scattering to assess homogeneity; pooled fractions consistently had a polydispersity of less than 15% and diameter of ~7.2–7.5 Å.

Travers T., López C.A., Van Q.N., Neale C., Tonelli M., Stephen A.G, & Gnanakaran S. (2018). Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain. Scientific Reports, 8, 8461.

+ Open protocol

+ Expand

Lipid Nanodisc Assembly Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Lipid nanodiscs were assembled as previously described.^{46-48 (link)} Briefly, MSP1D1 was expressed in BL21(DE3) bacterial cells and purified using Ni²⁺-affinity resin. The His₆-tagged protein was dialyzed into Tris-buffered saline (TBS) (20 mM tris; pH, 8; 100 mM NaCl; 0.5 mM EDTA), concentrated to 10 mg/mL (400 µM), snap-frozen in liquid N₂, and stored at −80°C. Lipids were prepared as an 80:20 molar ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (Avanti Polar Lipids), respectively, at a working concentration of 6 mM in TBS supplemented with 100 mM cholate. MSP1D1 and lipids were mixed at a 1:47 molar ratio, respectively, with 20 mM cholate and incubated for 1 hour at room temperature. To initiate the assembly of lipid nanodiscs, 0.5 mL of activated BioBeads (BioRad) were added to the sample and incubated for 2.5 hours. BioBeads were removed using a 0.22 µm spin filter, and nanodiscs were purified by size-exclusion chromatography (Superdex 200 Increase 10/300; GE Healthcare) with TBS as the running buffer. A single peak was obtained at 13 mL retention volume. Fractions were pooled, concentrated to 10 µM, and stored at 4°C.^{45,49 (link)}

Childers K.C., Peters S.C., Lollar P., Spencer H.T., Doering C.B, & Spiegel PC J.r. (2022). SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa. Blood Advances, 6(11), 3240-3254.

+ Open protocol

+ Expand

Reconstitution of MCU/EMRE Complex in Nanodiscs

Check if the same lab product or an alternative is used in the 5 most similar protocols

The detergent solubilized MCU/EMRE complex was reconstituted into nanodisc (Denisov, 2004 (link)) by combining the following components to a molar ratio of 1:4:10 (MCU/EMRE: MSP1: lipids (POPC:POPE:POPG, 3:1:1 molar ratio)). To remove detergents from the reaction, 0.4 g of BioBeads (BioRad) was added to the reaction and incubated with gentle agitation at 4 °C for 6 hours. BioBeads were replaced two additional times before the sample was further purified by gel filtration using a Superose 6 Increase 10/300 GL column (GE Healthcare) pre-equilibrated with Buffer A. The main peak – eluting around 14mL (Extended Data Fig. 3a and b) – was collected, concentrated to 1.3 mg/mL using an Amicon centrifugal concentrator (100kD cut-off, Millipore), and immediately used for cryo-EM grid preparation. The same procedure was used for the reconstitution of MCU alone into nanodiscs.

Wang Y., Nguyen N.X., She J., Zeng W., Yang Y., Bai X.C, & Jiang Y. (2019). Structural mechanism of EMRE-dependent gating of the human mitochondrial calcium uniporter. Cell, 177(5), 1252-1261.e13.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!