To investigate the effect of FHR1 on the cofactor activity of FH, the generation of C3b cleavage products by complement factor I (CFI) was assessed by SDS-PAGE and Western blotting. Two serially diluted isoforms of FHR1 (125 μg/ml-62.5 μg/ml), alone or in combination with FH (15.64 ng), were mixed with C3b (1.1 μg) and CFI (100 ng) and then incubated at 37°C for 30 min. After SDS-PAGE and Western blotting, the C3b degradation product (α’ 43 kDa) was detected with a C3c polyclonal antibody (Dako), followed by an HRP-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Labs). The membranes were developed with a chemiluminescent HRP substrate (Millipore), and the signals of the C3b degradation product (α’ 43 kDa) were analyzed by ImageJ.
Hrp conjugated anti rabbit igg antibody
Manufactured by Jackson ImmunoResearch
Sourced in Germany, United States
HRP-conjugated anti-rabbit IgG antibody is a secondary antibody that is conjugated with Horseradish Peroxidase (HRP). It is designed to bind to primary rabbit antibodies, allowing for the detection and visualization of target proteins in various immunoassays and immunochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime, by GE Healthcare (2 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
C3c polyclonal antibody, by Agilent Technologies (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Proteinase inhibitor cocktail, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
Ecl prime kit, by GE Healthcare (1 mentions)
Anti p stat6 antibody, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Vascular endothelial growth factor (vegf), by Immunotools (1 mentions)
Rabbit anti myc, by Cell Signaling Technology (1 mentions)
Hybond c extra nitrocellulose membrane, by Cytiva (1 mentions)
Amersham ecltm prime detection reagent, by GE Healthcare (1 mentions)
Imagequant las 3000, by GE Healthcare (1 mentions)
9 protocols using hrp conjugated anti rabbit igg antibody
1
FHR1 Impact on Complement Factor I Activity
2
Cas9 Protein Expression Analysis in Plant Tissues
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Cas9 expression was assessed via Western blot in leaf samples. Total protein samples were extracted from 4-weeks old green leaf tissue via grinding in liquid nitrogen with mortar and pestle and resuspended in protein loading buffer (Tris-HCl, pH:8.8) and heated to 100° for 5min. Extracts were centrifuged at 17,000x g for 5min at 25° and the supernatant was used for gel blot analysis. Protein were subject to electrophoresis in poly-acrylamide (0.375M Tris-HCl pH = 8.8, 8% Acrylamide, 0.05% APS, 0.1% SDS) matrix and transferred to PVDF membranes (Thermo Scientific, Cat#88520) with 70V under 4° for 90min. After blocking in milk solution, the membranes were incubated with monoclonal rabbit anti-HA antibody (Cell Signaling Technology, mAb#3724, 1:4000 dilution) and monoclonal mouse anti-β-Actin antibody (Millipore Sigma, Cat#MAB1501). Secondary HRP conjugated anti-rabbit IgG antibody (Jackson Immuno Research Laboratories, Inc. Code#111-0350144) and dye conjugated (infrared) anti-mouse antibody (LiCor IRDye 800CW Goat anti-rabbit IgG P/N#925-32210) were used were used to detect Cas9-HA and beta-actin, respectively. ECL (Bio-Rad, Cat#1705060) chemiluminescence and infrared fluorescence were imaged with Bio-Rad ChemiDoc MP system and analyzed with Bio-Rad Image Lab software.
3
Western Blot Quantification Protocol
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Each cell line was collected and lysed with an NP‐40 lysis buffer (1% NP40, 10 mM Tris HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA). Equal amounts of protein samples (30 μg) were electrophoresed in 7% acrylamide gel before transferring them onto membranes. The membranes were incubated with each primary antibody (same as the antibodies used in IHC) diluted at 1:1000 in 0.5% skimmed milk in Tris Buffered Saline with Tween20 overnight at 4°C. Horseradish peroxidase (HRP)‐conjugated anti‐mouse IgG antibody (Merck Millipore, Darmstadt, Germany) or HRP‐conjugated anti‐rabbit IgG antibody (Jackson ImmunoResearch, West Grove, Pennsylvania) was used as a secondary antibody. The membranes were soaked in Western Lightning Plus‐ECL (PerkinElmer, Tokyo, Japan) to visualize the immunoreaction. Immunoreactive bands were quantified and analyzed using AMERSHAM Image Quant 800 (Global Life Sciences Technologies Japan, Tokyo, Japan). The membranes were reused with mouse monoclonal anti‐beta actin antibody (Sigma‐Aldrich Japan K.K., Tokyo, Japan) to verify equal loading of proteins in each lane.
4
Protein Phosphorylation Analysis by Western Blot
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Cells were washed with PBS and lysed in 1% sodium dodecyl sulfate (Sigma) with a proteinase inhibitor cocktail (GE Healthcare, Little Chalfont, England) and a phosphatase inhibitor cocktail (Roche Diagnostics, Tokyo, Japan). Samples were electrophoresed and transferred onto PVDF membranes (Millipore). After blocking with 10% bovine serum albumin (Wako, Osaka, Japan), the membrane was incubated with anti-p-STAT6 antibody (Cell Signaling Technology, Tokyo, Japan) or anti-GAPDH antibody (Cell Signaling Technology) then incubated with HRP-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA, United States). The signal was detected with an ECL prime kit (GE Healthcare, Little Chalfont, United Kingdom) and imaged using a LAS 4000 analyzer (FUJIFILM, Tokyo, Japan).
5
Snorc-ECD-his-myc Binding Assay
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Recombinant proteins FGF2 (SigmaeAldrich), PDGF (Immunotools, Germany), VEGF (Immunotools), Wif-1 (prepared in mouse 24 (link) ) and fibronectin (kindly provided by Klaus von der Mark 25 ) were spotted onto a nitrocellulose membrane, blocked with 2% BSA in TBS for 1 h and incubated for 1 h with Snorc-ECDhis-myc (2 mg/ml in TBS) at RT. After intense washing and repeated blocking with BSA, the membrane was incubated over night with a rabbit anti-myc antibody (SigmaeAldrich) to detect bound Snorc-ECD-his-myc. Bound primary antibody was visualized by incubation with a HRP-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories) followed by ECL-based chemiluminescence detection using 2.5 mM Luminol, 0.4 mM pcoumaric acid, 100 mM TriseHCl, pH 8,5, 0.01% H 2 O 2 .
6
Immunoblotting Protein Analysis Protocol
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For immunoblotting, protein lysates or immunoprecipitates were denatured by boiling in Laemmli sample buffer, fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. Blots were blocked for 1 h at room temperature with 5% nonfat dry milk in TBS with 0.1% Tween 20 (TBST). Primary antibodies diluted in 5% BSA in TBST were added to the membranes and incubated overnight at 4°C with rocking. Primary antibodies consisted of rabbit anti-Myc (1:2,000; Cell Signaling; 71D10), rabbit anti-RHOT1 antibody (1:1,000; Aviva Systems Biology; ARP44817_P050), rabbit anti-HDAC6 antibody (1:2,000; Cell Signaling; 7558), rabbit anti-ERK1 (1:5,000; Abcam; ab32537), and affinity-purified custom rabbit anti–Miro1-AcK105 (1:2,500; Twiss laboratory, see above) antibodies. For the anti–Miro1-AcK105 antibody, preimmune and test-bleed sera were used at 1:1,000 dilutions to initially evaluate for specificity. After washing in TBST, blots were incubated with HRP-conjugated anti-rabbit IgG antibodies (1:5,000; Jackson Immunoresearch) diluted in blocking buffer for 1 h at room temperature. Signals were detected using ECL Prime (GE Healthcare) after extensive washing.
7
Quantifying Placental EV Proteins
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Total protein from placental EVs were extracted using RIPA buffer (50 mM Tris, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Nonidet P40 substitute, 1 mM PMSF, pH7.4) supplemented with protease inhibitor (Roche) and resolved by SDS-PAGE. Proteins were transferred onto HybondTM-C extra nitrocellulose membranes (Amersham Biosciences), which were blocked of non-specific binding with 5% non-fat milk powder/PBST. Membranes were then probed with rabbit anti-human complex IV (Thermo Fisher Scientific, A-6404, 1:750) or rabbit anti-human lamin B (Abcam, ab16048, 1:500) antibodies before applying HRP-conjugated anti-rabbit IgG antibodies (Jackson ImmunoResearch, 1:2000). Target proteins were visualised using AmershamTM ECLTM Prime detection reagent on an Image Quant LAS3000 (GE Healthcare) and images were annotated using Adobe® Photoshop® Elements 5.0.
8
AKT Phosphorylation Assay by Western Blot
9
Immunoblotting Protein Detection Protocol
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For immunoblotting, protein lysates or immunoprecipitates were denatured by boiling in Laemmle sample buffer, fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. Blots were blocked for 1 h at room temperature with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for anti-tagBFP, -GAPDH and -G3BP1 antibodies; 5% BSA in TBST was used for blocking anti-G3BP1PS149 antibody. Primary antibodies diluted in appropriate blocking buffer were added to the membranes and incubated overnight incubation at 4 °C with rocking. Primary antibodies consisted of: rabbit anti-G3BP1 (1:2000; Sigma), rabbit anti-G3BP1PS149 (1:1000; Sigma), rabbit anti-TagBFP (1:2000; Evrogen), and rabbit anti-GAPDH (1;2,000; CST). After washing in TBST, blots were incubated HRP-conjugated anti-rabbit IgG antibodies (1:5000; Jackson lab) diluted in blocking buffer for 1 h at room temperature. After washing signals were detected using ECL PrimeTM(GE Healthcare).
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