EVs were concentrated in Vivaspin 2, 5 kDa, concentrator columns to 20 µL and lysed using 10X RIPA lysis buffer containing protease inhibitor cocktail III (1:100; Thermo Fisher). Lysates from cells or EVs (20 µg) were loaded into a 4–12% Bis–Tris NUPAGE gel (Invitrogen) either under reducing (with DTT; GM130) or non-reducing (Alix, TSG101, CD9 and CD81) conditions and run at 125 V for 90 min in NUPAGE MES SDS buffer. Gels were subject to a wet transfer onto a nitrocellulose membrane using NUPAGE transfer buffer (15% methanol) at 150 V for 2 h at 4 °C. Membranes were blocked in 5% (w/v) milk in 0.1% (v/v) PBST (PBS Tween-20) for 1 h. Membranes were probed with antibodies against: TSG101 (Abcam; ab83, mouse, 1:500) Alix (Abcam; Ab117600, mouse, 1:2500), CD9 (Cambridge Biosciences / System Biosciences; EXOAB-CD9A-1; rabbit 1:1000), CD81 (Abcam; ab79559, rabbit, 1:1000), and GM130 (Abcam; ab52649, rabbit, 1:1000). The membranes were then washed three times with PBST then probed in milk buffer with either anti-Rabbit conjugated horseradish peroxidase (HRP) (Promega; w4011, 1:5,000) or anti-mouse HRP conjugated secondary antibody (Promega; w402B, 1:20,000). The membranes were washed again three times with PBST, then developed using ECL reagent (Amersham; Pico) for 5 min (RT) and imaged using a Bio-Rad Gel Doc XR +.
Anti mouse hrp conjugated secondary antibody
Manufactured by Promega
Sourced in United States
The Anti-mouse HRP conjugated secondary antibody is a laboratory reagent used to detect and visualize target proteins in Western blotting and other immunoassay applications. It is a secondary antibody that binds to mouse primary antibodies and is conjugated with the enzyme Horseradish Peroxidase (HRP), which catalyzes a colorimetric or chemiluminescent reaction for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Ab79559, by Abcam (1 mentions)
Ab52649, by Abcam (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Protease inhibitor cocktail 3, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Supersignal elisa femto substrate, by Thermo Fisher Scientific (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Promega (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Trans blot, by Bio-Rad (1 mentions)
Ff26a f9, by BioLegend (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Chemiluminescence kit, by Roche (1 mentions)
Slot blot apparatus, by Bio-Rad (1 mentions)
Hrp conjugated anti mouse secondary antibody, by Abcam (1 mentions)
6 protocols using anti mouse hrp conjugated secondary antibody
1
Extracellular Vesicle Protein Analysis
2
H. pylori-Induced Signaling Pathways
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AGS cells, human, and murine organoids co-cultured with H. pylori were and protein lysates were separated using 6% (AGS cells) or 10% (organoids) SDS PAGE mini gels, transferred to PVDF membranes, and membranes were blocked with BSA or milk as denoted. For detection of proteins, membranes were incubated overnight with anti-CagA rabbit antibody (Austral Biologicals; 1:5000, BSA), anti-pY99 antibody mouse antibody (Santa Cruz; 1:5000, BSA), anti-phospho-IRF-3 rabbit antibody #29047 (Cell Signaling Technology; 1:1000, BSA), anti-IRF-3 rabbit antibody #11904 (Cell Signaling Technology; 1:1000, BSA), anti-phospho-TBK1/NAK rabbit antibody #5483 (Cell Signaling Technology; 1:1000, BSA), anti-TBK1/NAK rabbit antibody #38066 (Cell Signaling Technology; 1:1000, BSA), anti-LC3A/B rabbit antibody #4108 (Cell Signaling Technology; 1:1000, BSA), anti-TMEM173/STING rabbit antibody #19851-1-AP (Proteintech; 1:1000, BSA), anti-GAPDH mouse antibody #MAB374 (Millipore Sigma; 1:5000, BSA), or anti-TRIM30 rabbit antibody #NBP2-41087 (Novus Biologicals; 1:1000, milk). An anti-rabbit or anti-mouse HRP-conjugated secondary antibody (Promega; 1:10000) was then incubated with membranes for 1 hour. Protein intensities were quantified using ImageJ software (NIH).
3
Phosphorylation Dynamics in Response to PAHs
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Cells were cultured in 96 well, white-walled plates and treated with the appropriate PAH for 24 h. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1%Triton X-100. Endogenous peroxides were quenched with 1% H2O2. Primary antibodies against phospho-p53 (Ser15) and phospho-H2AX (Ser139) were obtained from Cell Signaling (Beverly, MA) and used at a 1:500 dilution. Anti-mouse HRP-conjugated secondary antibody was purchased from Promega Corporation. Chemiluminescence was generated using a 1:1 reagent mixture of SuperSignal™ ELISA Femto Substrate (ThermoFisher Scientific) and normalized to DNA content using Hoecht florescence (Sigma-Aldrich, St. Louis, MO). Signal was quantified on a BioTek Synergy 2 plate reader (BioTek, Winooski, VT).
4
Detecting Tomato Virus Proteins
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Healthy and ToBRFV-, TMV- or ToMV-infected tomato leaves were ground in liquid nitrogen using a mortar and a pestle. The plant material was solubilized in 4 mL per g of RIPA buffer (10 mM Tris-HCl pH = 7.5; 150 mM NaCl; 0.5 mM EDTA; 0.1% SDS; 1% Triton X-100; 1% Deoxycholate) and mixed thoroughly. The homogenate was centrifuged at 3000× g for 15 min at 4 °C, then the supernatant was transferred to a new tube and centrifuged at 12,000× g for 15 min at 4 °C. The extracts were kept at −20 °C. The proteins were resolved in 15% SDS-PAGE gels and blotted onto nitrocellulose membranes (GE Healthcare) using a Trans-Blot (Bio-Rad, Hercules, CA, USA). After blocking, the membranes were incubated with a 1/250 dilution of antisera from immunized mice against the ToBRFV CP peptide or with 1/2 hybridoma supernatant dilutions. Next, an HRP-conjugated anti-mouse secondary antibody (Promega, Madison, WI, USA) was used at a 1/2500 dilution. As a control, a commercial ToBRFV antibody (DSMZ, Leibniz, Germany) was used at 1/1000 dilution and detected with an HRP-conjugated anti-rabbit secondary antibody (Promega) at 1/2500 dilution. The membranes were developed using SuperSignal West Pico PLUS (ThermoFisher, Hampton, NH, USA) and an Amersham Imager 600 (GE Healthcare Life Sciences, Wauwatosa, WI, USA).
5
Quantification of RelA expression
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Cells from monocyte subsets (sorted by FACS) were lysed in RIPA buffer (Sigma) containing 1 × protease inhibitors (Roche). Lysates were loaded onto 10% SDS-polyacrylamide gels (3 × 105 cells per lane). After transfer, the membranes were probed with anti-RelA, dilution 1:200, [532301] (#MAB5078; R&D Systems) or anti-GAPDH, dilution 1:2000, [FF26A/F9] (#649201; Biolegend), followed by HRP-conjugated anti-mouse secondary antibody, dilution 1:5000, (#W4021, Promega). Detection was performed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the protein band sizes were quantified using Image J, normalized to GAPDH.
6
Quantifying 5-Methylcytosine Levels in Genomic DNA
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