Click-iT protein labelling (Invitrogen) was performed according to the manufacturer’s instruction. Cells were incubated for 1 h in methionine-free medium. Afterwards, 50 µM AHA were added to the media for 3 h. The signal was detected using an HRP-conjugated streptavidin antibody (ab7403, Abcam, 1:20,000).
Ab7403
Manufactured by Abcam
Sourced in United Kingdom
Ab7403 is a laboratory equipment product offered by Abcam. It is designed to perform a specific function in a laboratory setting. The core function of this product is to [FUNCTION DESCRIPTION]. No further details or interpretations about its intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Mini protean tgx, by Bio-Rad (4 mentions)
Ab9110, by Abcam (3 mentions)
G 21234, by Thermo Fisher Scientific (3 mentions)
A9046, by Merck Group (3 mentions)
Chemidoc mp system, by Bio-Rad (3 mentions)
Click it protein labelling, by Thermo Fisher Scientific (2 mentions)
H6908, by Merck Group (2 mentions)
Ab6720, by Abcam (1 mentions)
Ab215166, by Abcam (1 mentions)
Ab243887, by Abcam (1 mentions)
Cresyl violet, by Merck Group (1 mentions)
Metal enhanced dab substrate kit, by Thermo Fisher Scientific (1 mentions)
Dpx mounting solution, by Merck Group (1 mentions)
Dot blot apparatus, by Bio-Rad (1 mentions)
Luminol enhancer solution, by Promega (1 mentions)
Ab6788, by Abcam (1 mentions)
Ab50279, by Abcam (1 mentions)
Ab5628, by Abcam (1 mentions)
Ab24170, by Merck Group (1 mentions)
30 protocols using ab7403
1
Click-iT Protein Labeling Protocol
2
Glycan-Based ELISA for Virus Receptor Binding
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Two biotinylated glycans were used in this assay, Neu5Acα2-3Galβ1-4GlcNAcβ-PAA-biotin (3′ SLN) and Neu5Acα2-6Galβ1-4GlcNAcβ-PAA-biotin (6′ SLN) (GlycoTech Corporation, USA). Receptor-binding specificity was analyzed by a solid-phase direct-binding assay as described previously (Shi et al. 2013 (link); Guo et al. 2018 ), with some modifications. Briefly, a 96-Well High-Binding Flat-Bottom Microplate (Corning, USA) was incubated with different concentrations (2-fold serial dilutions starting from the 256 HA titer) of viruses at 4 °C overnight; PBS was used as a negative control. After removal of the virus supernatant, the plates were washed four times with ice-cold PBS containing 0.1 per cent Tween 20 (PBST) and blocked with 0.2 ml of PBS containing 5 per cent bovine serum albumin (BSA) at room temperature for 2 h. After washing with ice-cold PBST, the plates were incubated with PBS containing either the 3′ SLN or 6′ SLN glycan (100 μl of 0.24 μM) at 4 °C overnight. After washing with PBST, the plate was incubated at room temperature for 1 h with horseradish peroxidase-conjugated streptavidin (ab7403, Abcam, UK). The plate was then incubated with 200 μl of a tetramethylbenzidine (TMB; Sigma) solution for 15 min at room temperature. Finally, the reaction was stopped with 100 μl of 0.5 M H2SO4. Absorbance was determined at 450 nm using a plate reader.
3
Immunohistochemical Analysis of Immune Markers
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Mouse tumor samples were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue slices were deparaffinized, rehydrated, and antigen retrieval was performed using 10 mM citrate buffer (pH 6.0). CD80 (polyclonal rabbit, 1:200, ab215166, Abcam), CD86 (monoclonal rabbit, 1:200, ab243887, Abcam), and CD69 (polyclonal rabbit, 1:250, A00529-2, Boster) were stained overnight at 4 °C. The samples were washed and then incubated with goat anti-rabbit biotinylated secondary antibody (1:1000, ab6720, Abcam) and visualized using a horseradish peroxidase (HRP)-conjugated ABC system (1:10,000, ab7403, Abcam).
4
Biotinylation Analysis of Transfected Cells
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To analyze the global biotinylation upon transfection with various mRNAs, CTV-1 cells were transfected as described above. Three hours after transfection, the transfected cells were supplemented with 50 µM biotin or not and cells were collected and lysed 5 h after biotinylation. Whole-cell lysates were separated by SDS-PAGE and were blotted as described above. Membranes were blocked in 1% BSA in PBS with 0.2% Triton X-100 and incubated in the same buffer with horseradish peroxidase-conjugated streptavidin (ab7403, Abcam, 1:10,000) overnight. After three quick washes with PBS, membranes were agitated for 5 min in 10% BSA in PBS with 1% Triton X-100 and biotinylated proteins were detected after three additional washes with PBS.
5
Immunohistochemical Analysis of ATP6AP2 in Mouse Placental Tissue
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EA 18 mouse placental tissue sections were de-paraffinized and rehydrated as described above. Antigen retrieval was performed using citrate buffer at 90–100°C (0.1 M trisodium citrate in MilliQ-Water, pH 6.0) for 25 min. Slides were washed in PBS-T before blocking in 1% BSA in PBS for 1 h at RT. To reduce non-specific background staining, slides underwent a 3% H2O2 (CSA Scientific, Gillman AUS) block for 30 min at RT. After which, slides were incubated with ATP6AP2 primary antibody (1 μg/mL; RnD Systems; AF5716, Minneapolis United States) in 1% BSA/PBS overnight at 4°C., before being incubated with a 1:300 dilution of anti-goat secondary antibody (HAF017, R&D systems) in 1% BSA/PBS for 1 h at RT. Tertiary incubation of streptavidin-biotin-horseradish peroxidase complex (ab7403, Abcam) was then carried out for 1 h at RT before immunostaining was developed by incubation in 3,3′-diaminobenzidine tetrahydrochloride solution (Metal Enhanced DAB Substrate Kit; ThermoFisher Scientific) for 10 min (Santa Cruz, sc-209686B, California United States). Cresyl violet (Sigma-Aldrich) counterstaining was applied to enhance tissue contrast before slides were cover slipped using DPX mounting solution (Sigma-Aldrich). Slides were then imaged through an Aperio slide imager at 40x magnification (Leica biosystems, Melbourne AUS).
6
Quantification of Xanthan Gum Binding
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Particles were prepared as above, using DMEM (1 ml) containing 0.04–20 μg of xanthan gum. Particles were treated with EDTA, and the solution (20 μl) was transferred to polyvinylidene difluoride (PVDF) membranes using a dot blot apparatus (Bio-Rad). PVDF membranes were incubated into a blocking solution of phosphate buffered saline (PBS) containing 0.05% (v/v) Tween 20 and 3% (w/v) BSA at room temperature for 2 hrs, and probed with 15 ml of PBS containing 0.2 μg of biotinylated wheat germ agglutinin (WGA; Sigma) at room temperature for 1 hr. After two washing steps using 20 ml of blocking solution containing 1% (w/v) of BSA for 5 min, the PVDF membranes were incubated with 15 ml of blocking buffer containing streptavidin-horseradish peroxidase (HRP) antibody (ab7403, Abcam; 1:25,000) at room temperature with gentle agitation for 30 min. Membranes were washed four times with 20 ml of blocking solution. Signal was revealed with 1 ml of luminol enhancer solution (Promega) based on instructions provided by the manufacturer. Signal was revealed using X-ray radiographic films.
7
Mycobacterium tuberculosis Serological Assay
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Serum samples were collected before and at weeks 3, 8, and 10 after M. tuberculosis infection. Microtiter 96-well plates (Costar) were incubated overnight with 5 μg/mL M. tuberculosis culture filtrate (CFP; BEI Resources) or 5 μg/mL whole-cell lysate (WCL; BEI Resources) in carbonate buffer at room temperature. Plates were then blocked for 2 hours with 3% BSA in PBS, and serially diluted serum samples were incubated for 45 minutes at 37°C. Plates were washed, and biotinylated polyclonal goat anti-mouse IgG2c (1:10,000; Abcam, catalog ab97253), goat polyclonal anti-mouse IgG1 (1:50,000; Abcam, catalog ab97238), or goat polyclonal anti-mouse IgG (1:100,000; Abcam, catalog ab6788) was added for 1 hour at room temperature. After incubation with streptavidin-HRP (1:30,000; Abcam, catalog ab7403) for 30 minutes at room temperature, binding was visualized by the addition of tetramethyl benzene in phosphate citric buffer (Sigma-Aldrich). The reaction was stopped with the addition of H2SO4, and absorbances were measured at 450 nm by a Tecan plate reader. Endpoint titers were estimated by the 5-polynomial sigmoidal curve of each sample interpolated with the threshold of the average of the negative controls ± 3 standard deviations.
8
Immunoblot Analysis of Cell Lysates
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To analyze total cell lysates by immunoblot, 1.2 × 106 cells were lysed in SDS–PAGE sample buffer, boiled for 5 min, and sonicated to shear DNA. Proteins were separated on 4–20% gradient gels (Mini-PROTEAN TGX; Bio-Rad, Hercules, CA) and transferred to nitrocellulose membrane (Bio-Rad). After blocking with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, the membrane was incubated with appropriate primary antibodies: chicken anti-BioID2 (1:5000; BID2-CP-100; BioFront Technologies) or rabbit polyclonal anti-hemagglutinin (1:2000; Ab9110; Abcam). The primary antibodies were detected using horseradish peroxidase (HRP)–conjugated anti-chicken (1:40,000; A9046; Sigma-Aldrich) or anti-rabbit (1:40,000; G21234; Thermo Fisher Scientific). The signals from antibodies were detected using enhanced chemiluminescence via a Bio-Rad ChemiDoc MP System (Bio-Rad, Hercules, CA). Following detection of each antibody, the membrane was quenched with 30% H2O2 for 30 minutes. To detect biotinylated proteins, the membrane was incubated with HRP-conjugated streptavidin (1:40,000; ab7403; Abcam) in 0.2% Triton X-100 in PBS for 45 min.
9
ADPr-Histone H3 Ubiquitylation Assay
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2 μg of H3 S10-ADPr peptide or a control unmodified H3 peptide was mixed with 1 μM DTX3L RING-DTC, 0.5 μM UBE1, 2.5 μM UBCH5A and 10 μM Ub in 50 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT and 1 mM ATP. After incubation at 37°C for 30 min, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue, and analyzed by western blotting. For ARH3 treatment, 2 μM ARH3 WT or ARH3 D77/78N were added to the reactions, and further incubated for 30 min at 37°C. Western blotting using anti-Ub (sc-8017, Insight Biotechnology) and anti-biotin (ab7403, Abcam) antibodies was performed to probe the ubiquitylated products.
10
Immunoblotting and Immunofluorescence Antibodies
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Following primary antibodies were used for this study: rabbit anti‐nucleolin (Abcam, ab50279, 1:1,000 for WB), rabbit anti‐nucleolin (ProteinTech, 10556‐1‐AP, 1:100 for IF), rabbit anti‐Kif5a (Abcam, ab5628, 1:1,000 for WB, 1:100 for Wes), rabbit anti‐HA (Sigma, H6908, 1:1,000 for WB), rabbit anti‐LAMP1 (Abcam, ab24170, 1:1,000 for WB), mouse anti‐GAPDH (Millipore, MAB374, 1:5,000 for WB), mouse anti‐β‐III tubulin (R&D systems, MAB1195, 1:1,000 for WB), mouse β‐III tubulin (Tuj1) (BioLegend 801202, 1:500 for IF), mouse anti‐NFH (Developmental Studies Hybridoma Bank, RT97, 1:200 for IF), chicken anti‐NFH (Abcam, ab72996, 1:1,000 for IF), mouse anti‐neurofilament (BioLegend 837904, 1:1,000 for IF), and sheep anti‐digoxigenin (Roche, 11207733910, 1:2,000 for ELISA). HRP‐conjugated secondary antibodies for immunoblots were purchased from Bio‐Rad Laboratories, Alexa Fluor Secondary antibodies were purchased from Jackson ImmunoResearch, and streptavidin‐HRP was purchased from Abcam (ab7403, 1:10,000).
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