Rabbit polyclonal anti-LARP4 antibody (16529-1-AP) was obtained from Proteintech (China). Mouse monoclonal anti-GFP antibody (MA5-15256) was purchased from Thermo Fisher. Rabbit anti-GFP antibody (AE011) was acquired from Abclonal (Wuhan, China). Goat anti-mouse IgG (H + L)-horseradish peroxidase (HRP) (172-1011) and Goat anti-rabbit IgG (H + L)-HRP (172-1019) were from BioRad. Mouse monoclonal anti-FLNA (sc-71118) was from Santa Cruz. Mouse monoclonal anti-GAPDH antibody (MA1-16757) and mouse monoclonal anti-6X His antibody (MA1-21315) were purchased from Thermo Fisher. Mouse monoclonal anti-β-Actin antibody (A00702-100) was purchased from GenScript. Alexa Fluor Plus 488 and 594, and Hoechst 33,342 were purchased from Thermo Fisher Scientific. Glutathione-Sepharose (L00206) was purchased from GE Healthcare. Ni-NTA resin (L00250-25) was obtained from Genscript. Streptavidin (Z02043-5, Genscript) was immobilized on NHS-activated Sepharose 4 Fast Flow beads (GE Healthcare) at 2 mg Streptavidin/1 mL beads according to the manufactures’ protocol.
Nhs activated sepharose 4 fast flow beads
Manufactured by GE Healthcare
Sourced in United States
NHS-activated Sepharose 4 fast flow beads are a type of agarose-based resin commonly used in affinity chromatography for the immobilization of various biomolecules. The beads are activated with N-hydroxysuccinimide (NHS), which allows for the covalent coupling of ligands containing primary amine groups. This makes the beads suitable for the purification and isolation of proteins, enzymes, and other molecules of interest.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 114, by Merck Group (3 mentions)
Thioglycollate medium, by BD (3 mentions)
Imidazole, by Merck Group (2 mentions)
Megatran 1.0 transfection reagent, by OriGene (2 mentions)
Fitc antibody labeling kit, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
Dynasore, by Bio-Techne (1 mentions)
Drabkin s reagent, by Merck Group (1 mentions)
Non immune mouse igg, by Merck Group (1 mentions)
Human m csf, by Merck Group (1 mentions)
Tacs mtt cell proliferation assay kit, by Bio-Techne (1 mentions)
Carbenicillin, by Thermo Fisher Scientific (1 mentions)
Redextract n amp tissue pcr kit, by Merck Group (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Protein a g agarose, by Thermo Fisher Scientific (1 mentions)
8 protocols using nhs activated sepharose 4 fast flow beads
1
Antibody Reagents for Protein Detection
2
Haptoglobin β Affinity Capture of HMGB1 in Sepsis
3
Haptoglobin Purification and Characterization
4
Human Innate Immune Receptor Interactions
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Human TLR4–MD-2 complex, human MD-2, TLR2, and soluble RAGE were obtained from R&D Systems. LPS (Escherichia coli; 0111:B4), APAP, Triton X-114, PGN from Bacillus subtilis, blasticidin S, NaSH, mouse IgG, and human macrophage-CSF (M-CSF) were purchased from Sigma-Aldrich. Protein A/G agarose and isopropyl-d -thiogalactopyranoside (IPTG) were from Thermo Fisher Scientific. NHS-activated Sepharose 4 fast flow beads were obtained from GE Healthcare. Thioglycollate medium was purchased from BD. Ultrapure LPS, Poly I:C, and type B CpG oligonucleotide were obtained from InvivoGen. Human S100 A12 was from Circulex Co. Anti–human and –mouse MD-2 antibodies were obtained from Imgenex. Anti-CBP tag antibody was from GenScript. Anti-p50 antibody (E381) and anti-p65 antibody were obtained from Epitomics and Santa Cruz Biotechnology, Inc., respectively. Serum ALT and AST levels were determined by color endpoint assay kits from BIOO Scientific.
5
Anti-PSA Nanobody Immobilization Protocol
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Custom-made anti-PSA
specific nanobodies
based on sequence N7 from Saerens et al.22 (link) (antigen-binding portion of the heavy chain from camelids, 0.49
mg/mL, PBS) were obtained from QVQ (Utrecht, The Netherlands) and
coupled to NHS activated Sepharose 4 Fast Flow-beads (GE Healthcare,
Little Chalfont, United Kingdom) according to the manufacturer’s
protocol. Briefly, 7 mL of 0.49 mg/mL anti-PSA in PBS was added to
14 mL of drained beads (1:1200 molar ratio; anti-PSA:NHS). The mixture
was incubated for 2 h at RT, the solution was spun down, supernatant
was removed, and the beads were resuspended in 50 mL of 0.1 M Tris-HCl
pH 8.5 and incubated for 2 h at RT. Immobilization of the nanobodies
was confirmed by analyzing the supernatant on a NuPAGE SDS-PAGE gel
(Thermo Fisher, Waltham, MA) with NuPAGE MOPS SDS running buffer (Thermo
Fisher) and after staining with Coomassie G-250 (SimplyBlue SafeStain,
Colloidal Blue Staining Kit, Thermo Fisher). Antibody beads were stored
as a 50% bead suspension (v/v) in 20% EtOH (20:80, EtOH:H2O, v/v)
at 4 °C. Before the beads were used, the 50% bead suspension
was washed with 1× PBS for ethanol removal and resuspended in
1× PBS to produce a 50% bead suspension (v/v).
specific nanobodies
based on sequence N7 from Saerens et al.22 (link) (antigen-binding portion of the heavy chain from camelids, 0.49
mg/mL, PBS) were obtained from QVQ (Utrecht, The Netherlands) and
coupled to NHS activated Sepharose 4 Fast Flow-beads (GE Healthcare,
Little Chalfont, United Kingdom) according to the manufacturer’s
protocol. Briefly, 7 mL of 0.49 mg/mL anti-PSA in PBS was added to
14 mL of drained beads (1:1200 molar ratio; anti-PSA:NHS). The mixture
was incubated for 2 h at RT, the solution was spun down, supernatant
was removed, and the beads were resuspended in 50 mL of 0.1 M Tris-HCl
pH 8.5 and incubated for 2 h at RT. Immobilization of the nanobodies
was confirmed by analyzing the supernatant on a NuPAGE SDS-PAGE gel
(Thermo Fisher, Waltham, MA) with NuPAGE MOPS SDS running buffer (Thermo
Fisher) and after staining with Coomassie G-250 (SimplyBlue SafeStain,
Colloidal Blue Staining Kit, Thermo Fisher). Antibody beads were stored
as a 50% bead suspension (v/v) in 20% EtOH (20:80, EtOH:H2O, v/v)
at 4 °C. Before the beads were used, the 50% bead suspension
was washed with 1× PBS for ethanol removal and resuspended in
1× PBS to produce a 50% bead suspension (v/v).
6
Antibody Staining and Purification Protocol
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In addition to antibodies that were previously described [20 (link),21 (link)] we used two commercial antibodies: mouse anti-MLH1 (IF 1:50, BD Biosciences, order number 551092) and rabbit anti-MLH1 (IF 1:50, Calbiochem, order number D00122409). We also used a chicken anti-SYCP3 antibody that was raised against a His-tagged version of a 99 amino acid-long (from 13E to 111E amino acids) peptide of SYCP3, which we overexpressed in Escherichia coli and purified using metal ion affinity chromatography. IgYs from the yolk of eggs of immunized chicken were extracted using a published protocol [82 ]. Anti-SYCP3 IgYs were affinity purified on immunizing-antigen coupled NHS-Activated Sepharose 4 Fast Flow beads (Cat#17-0906-01, Amersham, GE Healthcare) according to standard methods [83 ].
7
Kinase inhibitor protein profiling
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The two commercially available kinase inhibitors Purvalanol B (PurvB; Tocris, UK) and Bisindolylmaleimide-X (Bis-X; Enzo Life Sciences, USA) were tested in addition to 5 in-house investigational compounds designed to mimic kinase inhibitors referred to as L-1, L-2, L-3, L-4, and L-5 (previously SB6-060-05). The compounds L-1 and L-2 were prepared as shown in Supplementary Experimental Scheme 1 and accompanying experimental procedure. The compounds L-3, L-4, and L-5 were prepared as previously described (Bugge et al., 2016 (link); Blindheim et al., 2021a (link),b (link)). Kinase inhibitors containing carboxyl groups (PurvB, L-1, L-2, L-3, L-4, and L-5) were coupled to EAH Sepharose 4B beads (GE Healthcare, USA) and inhibitors with amino groups (Bis-X) were coupled to NHS-activated Sepharose 4 fast flow beads (GE Healthcare) according to manufacturer’s protocol.
Coupled beads (~75 μL) were added to Pierce™ spin-columns (Thermo Scientific; one column per kinase inhibitor). Bacterial extract (100 μg protein) was added to the column and incubated for 15 min at RT. The MIB assay was performed with on-column trypsinization as previously described (Petrovic et al., 2017 (link)).
Coupled beads (~75 μL) were added to Pierce™ spin-columns (Thermo Scientific; one column per kinase inhibitor). Bacterial extract (100 μg protein) was added to the column and incubated for 15 min at RT. The MIB assay was performed with on-column trypsinization as previously described (Petrovic et al., 2017 (link)).
8
Haptoglobin Purification and CD163 Expression
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