96-well plates (Maxisorp, Nunc) were coated with purified merozoites at 5 × 106 merozoites per well or recombinant merozoite surface antigens at 1–2 μg/ml24 (link). Briefly, plates were blocked with 1% casein, then incubated with serum samples (1/100–1/500) for 1 h, then washed with PBS-Tween 0.01%, followed by incubation for 30 min with purified C1q (10 μg/ml, Millipore) or normal serum (NS), heat-inactivated serum (HIS) from malaria naive donors, or C1q-depleted serum (Millipore) at 10% final concentration as a source of complement. After washing plates, C1q fixation was detected by incubating with rabbit anti-C1q antibodies (in-house) and then with anti-rabbit-IgG-HRP (Millipore), incubated for 1 h each. ABTS (Life Technologies) was added, incubated for 30 mins and absorbance quantified using a plate reader. All steps were conducted at room temperature (~21 °C). For studies of membrane attack complex (MAC) formation, normal serum was used as a complement source, and (rabbit-) anti-C5b/C9 antibodies (Millipore), and anti-rabbit-IgG-HRP (Millipore) were used for detection. IgG subclass ELISAs were done as previously described29 (link),46 (link),54 (link) using mouse anti-human IgG subclass monoclonal antibodies (Thermo Fischer Scientific) and anti-mouse-IgG HRP (Millipore) detection antibodies. (Detailed protocols are available from the authors on request).
Anti rabbit igg hrp
Manufactured by Merck Group
Sourced in United States, Germany
Anti-rabbit IgG-HRP is a laboratory reagent used for the detection and quantification of rabbit immunoglobulin G (IgG) in various immunoassay techniques. It consists of rabbit IgG antibodies conjugated with the enzyme horseradish peroxidase (HRP). This conjugate can be used as a secondary antibody to bind to and label rabbit primary antibodies, enabling their visualization and quantification through enzymatic color development or chemiluminescent reactions.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg hrp, by Merck Group (25 mentions)
Anti gapdh, by Santa Cruz Biotechnology (9 mentions)
Immobilon crescendo western hrp substrate, by Merck Group (9 mentions)
Amersham imager 600, by GE Healthcare (9 mentions)
Protease inhibitor, by Merck Group (7 mentions)
Anti mouse igg peroxidase, by Merck Group (7 mentions)
Anti mouse, by Merck Group (6 mentions)
Anti s2, by Sino Biological (5 mentions)
Anti s1, by Sino Biological (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Amersham hybond ecl, by GE Healthcare (3 mentions)
Image studio program, by LI COR (3 mentions)
Ecl prime, by Cytiva (3 mentions)
C digit western blot scanner, by LI COR (3 mentions)
Ecl western blot detection system, by Cytiva (3 mentions)
Phosphatase inhibitor cocktail solution, by GenDEPOT (3 mentions)
Anti flag beads, by Merck Group (3 mentions)
Anti mouse igg hrp, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
73 protocols using anti rabbit igg hrp
1
Measuring Complement Activation by Malaria Antigens
2
Protein Extraction and Western Blot Analysis
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Whole cells extract was isolated using RIPA buffer (NaCl 150 mM - Tris HCL pH7.35 50 mM - DOC 1% - NP40 1%) supplemented with protease inhibitor (Ref #04693116001, Roche). Concentration of isolated proteins was determined using Bradford assay (500-0006, Bio-Rad).
Western Blot analysis were performed using the following primary antibodies: anti-GATA3 (1/1000; Ozyme #5852), anti-PHOX2B (1/1000, Ozyme #PA-5115754), anti-c-JUN (1/1000, Cell Signaling #9165), anti-MAML2 (1/500, Sigma–Aldrich clone 4A1 # WH0084441M3), anti-RUNX1 (1/500, Santa Cruz SC-365644), anti-GAPDH (1/500, Sigma–Aldrich #G9545). Anti-mouse IgG HRP (1/10000, Sigma #A4416), anti-rabbit IgG HRP (1/10000, Sigma–Aldrich #A9169) and anti-goat IgG HRP (1/10000, Sigma #A5420) were used as secondary antibodies. Densitometric analyses of western blots were performed using Image J software.
Western Blot analysis were performed using the following primary antibodies: anti-GATA3 (1/1000; Ozyme #5852), anti-PHOX2B (1/1000, Ozyme #PA-5115754), anti-c-JUN (1/1000, Cell Signaling #9165), anti-MAML2 (1/500, Sigma–Aldrich clone 4A1 # WH0084441M3), anti-RUNX1 (1/500, Santa Cruz SC-365644), anti-GAPDH (1/500, Sigma–Aldrich #G9545). Anti-mouse IgG HRP (1/10000, Sigma #A4416), anti-rabbit IgG HRP (1/10000, Sigma–Aldrich #A9169) and anti-goat IgG HRP (1/10000, Sigma #A5420) were used as secondary antibodies. Densitometric analyses of western blots were performed using Image J software.
3
In Vitro Kinase Assay for BCAP Phosphorylation
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For the in vitro kinase assays, 2 μg of dephosphorylated BCAP (FL) or 100 μg of dephosphorylated myelin basic protein (31314; Active Motif) were diluted in 500 μl of kinase buffer (50 mm HEPES, 10 mm MgCl2, 0.01% BRIJ35, 1 mm EGTA, and 150 μm ATP, pH 7.5). Upon adding 60 pmol of SYK (PV3857; Thermo Fisher Scientific), LYN (PV6448; Thermo Fisher Scientific), BTK (PV3363; Thermo Fisher Scientific), TYK2 (PV4790; Thermo Fisher Scientific), ITK (PV4193; Thermo Fisher Scientific), CSNK1A1 (PV3850; Thermo Fisher Scientific), or CSNK2A1 (PV3248; Thermo Fisher Scientific), the samples were incubated at 30 °C for 30 min. The reaction was stopped using 4× SDS loading dye, and the samples were analyzed using Western blotting. For chemiluminescence detection, anti-BCAP (AF4857; R&D Systems), anti-phosphotyrosine (Ab179530; Abcam), anti-phosphoserine (Ab9332, Abcam), anti-rabbit IgG-HRP (A0545; Sigma), and anti-goat IgG-HRP (A5720; Sigma) were used.
4
Rspo1 Knockdown Protocol Using shRNA
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Short hairpin RNA (shRNA) directed against Rspo1 was purchased from Thermo (U.S.). The Rspo1 shRNA1 target sequence was 5′-TACACTTGGTGCAGAAGTT-3′, the Rspo1 shRNA2 target sequence was 5′-TGCACTTGTTCATGTCGGG-3′, and the nonsense shRNA sequence was 5′-TACGCATCCGCAACTGCAG-3′. The rabbit anti-Rspo1 antibodies used for Western blotting, immunoprecipitation and immunohistochemical assays were purchased from Abcam. The mouse anti-β-actin antibodies used for the immunoblotting assay were purchased from Sigma. The secondary antibodies anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Sigma.
5
Optimized Immunoblot Protein Quantification
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Extracted proteins for immune-based Western blotting were first separated, according to molecular weight, using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels, followed by electrotransfer to nitrocellulose membranes (Amersham Hybond-ECL, GE Healthcare, Chicago, IL, USA), as described before [26 (link)]. Equal amounts of protein and volume were loaded onto a 12.5% polyacrylamide gel for CYGB, heme oxygenase 1 (HO-1) and NF E2-related factor 2 (NRF2). Membranes were blocked in TBS-T (Tris-buffered saline; 0.1% Tween-20), containing 5% non-fat dry milk, for 1 h at room temperature. After blocking, membranes were incubated overnight at 4 °C with primary antibodies (anti-CYGB, Proteintech, Rosemont, IL, USA; 13317-1-AP; anti-β-2-Microglobulin (B2M), Proteintech, 13511-1-AP; anti-β-Actin (ACTB), Santa Cruz, sc-47778; anti-HO-1, Proteintech, 10701-1-AP; anti-NRF2, Proteintech, 16396-1-AP). The following day, membranes were washed with TBST-T and incubated for 1 h with horseradish-conjugated secondary antibodies (anti-rabbit IgG HRP, Sigma, GENA934-1ML; anti-mouse IgG HRP, Invitrogen, 31430). The signal was revealed using ECL Prime (Amersham, GERPN2232) on an Amersham Imager 680 (GE Life Sciences; Piscataway, NJ, USA) and exported and quantified using the Image Studio™ program (LI-COR Biosciences, Lincoln, NE, USA).
6
Western Blotting of Bacterial Proteins
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The cultures for Western blotting were grown in the indicated medium (±inducer), harvested, and resuspended in an appropriate volume of 2× Laemmli buffer (Sigma-Aldrich) according to the optical density at 600nm of the initial culture (OD600) followed by 15 min incubation at 95°C. The proteins were resolved by SDS-PAGE (15%) and transferred to a PVDF membrane (Merck Millipore) using a wet- or semi-dry transfer apparatus. After incubation with the blocking buffer (2.5% skimmed milk in 1× Tris-buffered saline with 0.1% Tween-20), one of the following primary antibodies was added: anti-QstR (raised in rabbits against synthetic peptides; Eurogentec #1412414) at 1:2500 dilution; anti-ComEA (raised in rabbits against synthetic peptides; Eurogentec #GP1248; (34 (link))), anti-Hcp (against synthetic peptide, Eurogentec #1510528; (35 (link))), anti-HapR (against synthetic peptide, Biomatik #A000542; (11 (link))), all at 1:5000 dilution; anti-RNAP-beta (Neoclone #WP001; raised in mouse) at dilution 1:2000. The secondary antibodies used were anti-rabbit IgG HRP (Sigma-Aldrich #A9169) and anti-mouse IgG HRP (Sigma-Aldrich #A5278), both at 1:10000 dilutions. The proteins were visualized by chemiluminescence using Lumi-LightPLUS Western Blotting Substrate (Roche).
7
Immune-Based Western Blotting Protocol
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Extracted proteins for immune-based western blotting were first separated, according to molecular weight, using sodium dodecyl sulphate polyacrylamide gel-electrophoresis (SDS-PAGE) gels, followed by electrotransfer to nitrocellulose membranes (Amersham Hybond-ECL, GE Healthcare) as described before [47 (link),48 ]. Equal amounts of protein and volume were loaded onto a 12.5% polyacrylamide gel for CYGB, heme oxygenase 1 (HO-1), and NF E2 related factor 2 (NRF2). Membranes were blocked in TBS-T (Tris-buffered Saline; 0.1% Tween-20), containing 5% non-fat dry milk, for 1 h at room temperature. After blocking, membranes were incubated overnight at 4 °C with primary antibodies (anti-CYGB, Proteintech, 13317-1-AP; anti-β-2-Microglobulin (B2M), Proteintech, 13511-1-AP; anti–HO–1, Proteintech, 10701-1-AP; anti-NRF2, Proteintech, 16396-1-AP). The following day, membranes were washed with TBST-T, and incubated during 1 h with horseradish-conjugated secondary antibodies (anti-rabbit IgG HRP, Sigma, GENA934-1 ML). The signal was revealed using ECL Prime (Amersham, GERPN2232) on a C-DiGit® Western blot scanner (LI-COR Biosciences) and exported and quantified using Image Studio™ program (LI-COR Biosciences).
8
Protein Detection in Cells and Spheroids
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In order to detect various proteins from cells and spheroids, a proper amount of samples were homogenized and digested in 1× RIPA buffer (Cell Signaling, Denver, MA, USA) which includes 1 mM PMSF (Fluka, Switzerland), 2 g/mL Aprotinin (Sigama, Steinheim, Germany), 1 mM DTT(Invitrogen, Carlsbad, CA, USA) and phosphatase inhibitor cocktail solution (GenDEPOT, Barker, TX, USA) on ice for 1 hr. After the digestion, samples were centrifuged for 25 min at 14,000 rpm in cold microcentrifuge. Then supernatants were collected for use. Western blot experiments were performed following the standard protocol. The following primary antibodies were purchased: anti-YAP/TAZ (8418, Cell Signaling) and anti-EpCAM (ab71916, Abcam). anti-EpCAM was used and for internal control, an anti-GAPDH antibody (2118, Cell Signaling) was used. Anti-rabbit IgG-HRP (A0545, Sigma) was used as the secondary antibody. Specific bands were detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
9
Polyclonal Anti-Cdc48 Antibody Detection
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Polyclonal anti-Cdc48 antibodies were a generous gift from T. Endo (Kyoto Sangyo University) and were diluted into TBS-T buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) at ∼1/20,000. An anti-HA antibody (#M180-3) was purchased from MBL and was diluted into TBS-T buffer at 1/10,000. Anti-mouse IgG-HRP (#A4416-1 ML) and anti-rabbit IgG-HRP (#A6154-1 ML) were obtained from Sigma-Aldrich, diluted into TBS-T buffer at 1/7,000, and used as secondary antibodies. A mono- and polyubiquitinated conjugated/mouse mAb horseradish peroxidase conjugate (FK2H) (#PW0150) was obtained from BioMol.
10
Quantifying SARS-CoV-2 Viral Proteins
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Lysate from HEK293T cells used to produce lentiviral vectors was collected through a 40-minute incubation on ice in RIPA lysis buffer (50mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, Nonidet P-40, 0.1% SDS) supplemented with protease inhibitor (Sigma, P8340). Samples were run on a 10% acrylamide SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with anti-S1 (Sino Biological, 40591-T62; RRID:AB_2893171), anti-S2 (Sino Biological, 40590; RRID:AB_2857932), anti-p24 (NIH HIV Reagent Program, ARP-1513), and anti-GAPDH (Santa Cruz, Cat# sc-47724, RRID: AB_627678). Secondary antibodies included Anti-Rabbit-IgG-HRP (Sigma, A9169; RRID:AB_258434) and Anti-Mouse (Sigma, Cat# A5278, RRID: AB_258232). Blots were imaged using Immobilon Crescendo Western HRP substrate (Millipore, WBLUR0500) and exposed on a GE Amersham Imager 600. Band intesnsities were quantified using NIH Image J analysis software (Bethesda, MD).
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