Sandwich ELISAs were performed as previously described [23 (link)]. Briefly, 96-well black NUNC Maxisorp flat bottom plates (Thermo Scientific, Waltham, MA, USA) were coated with 100 µL/well of 1 µg/mL of the capture antibody, Stx1-2 [24 (link)] and incubated at 4 °C overnight. After overnight incubation, the plates were washed two times with 0.02 M Tris buffered saline with 0.9 % NaCl, pH 7.4, and 0.05% Tween-20 (TBST), blocked in 5% non-fat dry milk (NFDM)-TBST for an hour at 37 °C, and then incubated with samples for 1 h at 37 °C. The plates were washed with TBST six times and incubated with 100 ng/mL of detection antibody, Stx1 pAb [23 (link)], in NFDM-TBST. Goat anti-rabbit HRP (Promega, Madison, WI, USA), 10 ng/mL in NFDM-TBST, was added as a secondary antibody after washing six times with TBST. Plates were developed with 100 µL SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA), and luminescence counts per second (CPS) were read on a Victor 3 plate reader (Perkin Elmer, Shelton, CT, USA) for 0.1 s. Each treatment was performed in triplicate and repeated twice on two different days.
Goat anti rabbit hrp
Manufactured by Promega
Sourced in United States
Goat anti-rabbit-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used to detect and quantify rabbit primary antibodies in various immunoassay techniques such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse hrp, by Promega (4 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Nunc maxisorp flat bottom plate, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Victor3 plate reader, by PerkinElmer (1 mentions)
Immobilon p 0.45 m pvdf membrane, by Merck Group (1 mentions)
Western lightening plus ecl kit, by PerkinElmer (1 mentions)
Gradient sds page gel, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Tyramide signal amplification kit, by Thermo Fisher Scientific (1 mentions)
Bs 3759r, by Bioss Antibodies (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Bz x700, by Keyence (1 mentions)
Trans blot apparatus, by Bio-Rad (1 mentions)
M6a antibody, by Abcam (1 mentions)
Mgme1, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
β actin, by Abcam (1 mentions)
Ab10436, by Abcam (1 mentions)
9 protocols using goat anti rabbit hrp
1
Sandwich ELISA for Stx1-2 Detection
2
Western Blot Protocol for CBX7 and DCAF12l1 Quantification
3
Colocalization of HAS2 and S100A4 in Fascia
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Stecco et al.44 (link) showed that HA-rich cells are derived from a family of fibroblasts and suggested that these cells are called fasciacytes, a new class of fascia-associated cells, with S100A4 expression as their characteristic.
Double immunofluorescence staining was performed using the tyramide signal amplification kit (Thermo Fisher, Waltham, MA) to detect colocalization of HAS2 and S100A4 in the fascia. All sections were subjected to the following protocols: The tissues were washed in PBS for 3 minutes. After incubation with 0.1% H2O2 and 1% bovine serum albumin, frozen tissue sections were incubated with anti-HAS2 (mouse, 1:200, Santa Cruz Laboratories) and anti-S100A4 (rabbit, 1:100, # BS-3759R, Bioss, Woburn, MA) overnight at 4°C, followed by goat anti-mouse-DyLight 594 (1:500, Abcam, Cambridge, United Kingdom) and goat anti-rabbit-HRP (1:100, Promega, Fitchburg, WI) for 2 hours at room temperature. For S100A4 staining, sections were subsequently incubated with Alexa Fluor 488-conjugated tyramide (1:200, Thermo Fisher) for 10 minutes and mounted with ProLong Gold Antifade reagent with DAPI (Thermo Fisher). Images were acquired with a fluorescence microscope BZ-X700 (Keyence, Osaka, Japan) equipped with a 40× objective lens.
Double immunofluorescence staining was performed using the tyramide signal amplification kit (Thermo Fisher, Waltham, MA) to detect colocalization of HAS2 and S100A4 in the fascia. All sections were subjected to the following protocols: The tissues were washed in PBS for 3 minutes. After incubation with 0.1% H2O2 and 1% bovine serum albumin, frozen tissue sections were incubated with anti-HAS2 (mouse, 1:200, Santa Cruz Laboratories) and anti-S100A4 (rabbit, 1:100, # BS-3759R, Bioss, Woburn, MA) overnight at 4°C, followed by goat anti-mouse-DyLight 594 (1:500, Abcam, Cambridge, United Kingdom) and goat anti-rabbit-HRP (1:100, Promega, Fitchburg, WI) for 2 hours at room temperature. For S100A4 staining, sections were subsequently incubated with Alexa Fluor 488-conjugated tyramide (1:200, Thermo Fisher) for 10 minutes and mounted with ProLong Gold Antifade reagent with DAPI (Thermo Fisher). Images were acquired with a fluorescence microscope BZ-X700 (Keyence, Osaka, Japan) equipped with a 40× objective lens.
4
Western Blotting of Immune Receptors
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Western blotting was performed as described in Bonnin et al. [2 (link)], with the following modifications. Proteins were transferred onto PVDF membranes using semi-dry Trans-Blot apparatus (Bio-Rad), anti-actin was from MP (clone C4, France; RRID:AB_2335127), anti-hTLR3 (clone D10F10; RRID:AB_10829166; MA, USA), anti-hTLR7 (clone D7; RRID:AB_10692895), anti-hTLR8 (clone D3Z6J; RRID:AB_2797755), anti-hTLR9 (clone D9M9H; RRID:AB_2798290), anti-RIG-I (clone D14G6; RRID:AB_2269233), anti-MDA5 (clone D74E4; RRID:AB_10694490) were from Cell Signaling, and secondary Goat anti-Rabbit HRP (RRID:AB_430833) conjugate and secondary Goat anti-Mouse HRP (RRID:AB_430834) conjugate were from Promega. Image acquisition and relative band intensity measurement were performed using a Chemi Doc XR+ apparatus (Bio-Rad) driven by Image Lab software version 6.1.
5
Quantifying m6A Abundance in mRNA
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To determine relative abundance of m6A using membrane antibody-based detection, 100 and 50 ng of poly(A+) selected mRNA (described above) was spotted on SensiBlot plus Nylon membrane (Fermentas #M1002) and air dried for 5 min. The membrane was UV crosslinked using Stratalinker and blocked with PBST-5% (w/v) milk for 3 h. Membrane was incubated overnight with m6A antibody (abcam#15320, 1:2500), washed and subsequently incubated with goat antirabbit-HRP (Promega, 1:10,000). The membrane was developed using Immobilon Femto Western HRP substrate (Thermo #34094). The intensity of the dot blots was measured by using Image J software.
6
Western Blot Analysis of Mitochondrial Proteins
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Primary antibodies used for western blotting were as follows: MGME1 (Sigma HPA040913, 1 : 500 dilution), β-actin (Sigma A2228, 1 : 300 000), PolgA (Santa Cruz, 1 : 1000), mtSSB1 (kindly donated by Dr Kang) and TFAM (kindly donated by Dr Wiesner). Secondary antibodies used were as follows: goat anti-rabbit HRP (Promega W401B, 1 : 2000) and goat anti-mouse HRP (Promega W402B, 1 : 2000).
7
Dicer Protein Quantification in Westerns
8
Immunofluorescence and Western Blot Analyses
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Localisation of proteins by immunofluorescence was carried out in fixed 143B cells as previously described (22 (link)). Images were captured using a Zeiss LSM 880 confocal microscope.
The following antibodies were used for immunofluorescence experiments in this work: mouse anti-TOM22 (Abcam, ab10436, 1:250), Alexafluor-594 anti-mouse (Molecular Probes, A11005, 1:1000), rabbit anti-TFAM (gifted by Prof. Rudolf Wiesner, 1:500), Alexafluor-405 anti-rabbit (Molecular Probes, A31553, 1:1000), rat anti-HA (Roche, 11867431001, 1:500), Alexafluor-488 anti-rat (Molecular Probes, A11006, 1:1000). Mounting medium used was either ProLong Gold Antifade Mountant (Molecular Probes), or ProLong Gold Antifade Mountant with DAPI (Molecular Probes).
For western blot analyses, ∼20 μg of extracted proteins were resolved on SDS-PAGE 4–12% bis-tris gels (Life Technologies). The following antibodies were used for western blotting in this work: mouse anti-FLAG (Sigma, F1804, 1:2000), rabbit anti-FLAG (Sigma, F7425, 1:2000), rat anti-HA (Roche, 11867431001, 1:1000), rabbit anti-Histone H4 (Abcam, ab10158, 1:5000), rabbit anti-SSB1 (kindly gifted by Prof D. Kang, 1:4000), mouse anti-TOM22 (Abcam, ab10436, 1:5000), mouse anti-GAPDH (Abcam, ab9484, 1:10 000), goat anti-rabbit HRP (Promega, W401B, 1:2000), goat anti-mouse HRP (Promega, W402B, 1:2000), goat anti-rat HRP (Santa Cruz, SC2065, 1:1000).
The following antibodies were used for immunofluorescence experiments in this work: mouse anti-TOM22 (Abcam, ab10436, 1:250), Alexafluor-594 anti-mouse (Molecular Probes, A11005, 1:1000), rabbit anti-TFAM (gifted by Prof. Rudolf Wiesner, 1:500), Alexafluor-405 anti-rabbit (Molecular Probes, A31553, 1:1000), rat anti-HA (Roche, 11867431001, 1:500), Alexafluor-488 anti-rat (Molecular Probes, A11006, 1:1000). Mounting medium used was either ProLong Gold Antifade Mountant (Molecular Probes), or ProLong Gold Antifade Mountant with DAPI (Molecular Probes).
For western blot analyses, ∼20 μg of extracted proteins were resolved on SDS-PAGE 4–12% bis-tris gels (Life Technologies). The following antibodies were used for western blotting in this work: mouse anti-FLAG (Sigma, F1804, 1:2000), rabbit anti-FLAG (Sigma, F7425, 1:2000), rat anti-HA (Roche, 11867431001, 1:1000), rabbit anti-Histone H4 (Abcam, ab10158, 1:5000), rabbit anti-SSB1 (kindly gifted by Prof D. Kang, 1:4000), mouse anti-TOM22 (Abcam, ab10436, 1:5000), mouse anti-GAPDH (Abcam, ab9484, 1:10 000), goat anti-rabbit HRP (Promega, W401B, 1:2000), goat anti-mouse HRP (Promega, W402B, 1:2000), goat anti-rat HRP (Santa Cruz, SC2065, 1:1000).
9
Western Blot Analysis of PNT1 and OPB Proteins
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1 x 10 7 cells lysed in NuPAGE sample buffer (supplemented with β-mercaptoethanol) were loaded on to a 4-12% NuPAGE Bis Tris Gel and run at 150V in MOPS buffer. The gels were transferred onto a PVDF membrane using the wet transfer system XCell II Blot Module of Invitrogen at a constant voltage of 30V for 90 min. The blot was blocked with 5% Skimmed Milk (Sigma) for 1 h. Primary antibodies were added at the following concentrations: rabbit polyclonal PNT1 antibody-1:500 overnight at 4°C, sheep polyclonal OPB antibody-1:20,000 1 h at room temperature. After 3 washes in 1X TBST, goat anti rabbit HRP (Promega) and donkey anti-sheep HRP (Santa Cruz) were added at 1;5000 dilution for 1 h. The blots were washed with 1X TBST and overlayed with Clarity Max substrate (BioRad). The blots were developed in a myECL Imager (Thermo Fisher Scientific).
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