Western blotting was performed as previously described [6 (link), 20 (link)]. For total protein extraction, cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. The primary antibodies were rabbit IgG anti-collagen I, V (Rockland), or IV (Bioss Antibodies); rabbit IgG anti-fibronectin (Proteintech, Chicago, IL); rabbit IgG anti-α-SMA (GeneTex, Irvine, CA); mouse IgG anti-nestin (Santa Cruz Biotechnology); rabbit IgG anti-phospho-SMAD3 (pS423 pS435) (Rockland, PA); rabbit IgG anti-SMAD2, SMAD3, and SMAD4 (Cell Signaling Technology, Danvers, MA); and rabbit IgG anti-RGC-32 [6 (link)]. The secondary antibodies were goat anti-rabbit or anti-mouse IgG-HRP (Santa Cruz Biotechnology). Immune complexes were detected using enhanced chemiluminescence (Denville Scientific Inc., Holliston, MA). The density of the bands was measured using UN-SCAN-IT software, version 7.1 (Silk Scientific Inc., Orem, UT).
Goat anti mouse or anti rabbit igg hrp
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
Goat anti-mouse or anti-rabbit IgG-HRP is a secondary antibody conjugated with Horseradish Peroxidase (HRP). It is used to detect and quantify the presence of mouse or rabbit primary antibodies in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Microbca kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Ecl system, by GE Healthcare (1 mentions)
Membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin monoclonal igg1 antibody, by Santa Cruz Biotechnology (1 mentions)
Alex fluor 488, by Thermo Fisher Scientific (1 mentions)
Texas red conjugated goat anti rabbit, by Rockland Immunochemicals (1 mentions)
Rabbit igg isotype control, by Southern Biotech (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Rabbit polyclonal antibody against human gapdh, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by MultiSciences Biotech (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by BestBio (1 mentions)
5 protocols using goat anti mouse or anti rabbit igg hrp
1
Western Blotting Protein Analysis Protocol
2
Evaluating Tight Junction Protein Expression
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IPEC-J2 cells were treated with DON in the absence or presence of pretreatment with TLR2 ligands, washed with PBS and lysed in a lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100), followed by a quantitation of protein using Micro BCA kit (Thermo, Rockford, USA). For isolation of cytosolic and membrane parts from IPEC-J2 cells, membrane protein extraction kit (Thermo) was used by its instruction. As previously described [17 (link)], the same amount of protein extracts was loaded in 10% Tris–glycine polyacrylamide gels and electrophoresed. Then, the proteins were transferred onto a polyvinylidene difluoride (PVDF) microporous membrane for 2 h at 4 °C and blocked with 5% skim milk in TBS-T (20 mM Tris HCl, 100 mM NaCl, 0.05% Tween 20) for 90 min. The blot was incubated with rabbit anti-claudin-3, -occludin or -zonula occludens (ZO)-1 antibodies (Invitrogen), anti-p-AKT, -p-P70S6K, -Akt, -FAK, and -Bcl-2 antibodies (Cell signaling), or mouse anti-β-actin monoclonal IgG1 antibody (Santa Cruz Biotechnology, Grand Island, USA) overnight. Subsequently, the membrane was washed and incubated with goat anti-rabbit or anti-mouse IgG-HRP (Santa Cruz Biotechnology) for 1 h. The target protein was visualized with enhanced chemiluminescence (ECL) system (GE Healthcare, Waukesha, USA), followed by analysis using ChemiDoc XRS (Bio-rad, Hercules, USA).
3
Antibody-Based Characterization of HA-Tagged IFI44
4
Western Blot Analysis of Membrane Proteins
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10 μl of membrane fractions were denaturated at 95°C for 10 min in Laemmli buffer, size fractionated by SDS/PAGE, transferred to PVDF membranes (Immobilon-P; Merck Millipore, Darmstadt, Germany) by semi-dry blotting and detected by chemiluminescence (PierceTM ECL Western Blotting Substrate, Thermo Fisher Scientific Inc.).
Membranes were incubated for 1.5 h with the appropriate antibody diluted according to manufacturer's instructions in TBST containing 6.5% non-fat dry milk. Membranes were washed three times for 5 min with TBST and, when required, incubated with the secondary antibody for another 1.5 h. Primary antibodies used were rabbit anti-GFP-HRP (Santa Cruz Biotechnology, Inc.), rabbit anti-DMP1 (Thermo Fisher Scientific Inc.), rabbit anti-BiP2 (Agrisera) for detection of ER-microsomes, rabbit anti-H+-ATPase (Agrisera) for detection of PM-derived vesicles and rabbit anti-v-ATPase (Agrisera) for detection of tonoplast-derived vesicles. Goat anti-mouse or anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Inc) were used as secondary antibodies. Custom anti-DMP1 primary antibodies were raised against the DMP1 C-terminal peptide KRSGIGYAPIAEEVGAE corresponding to aa 181 to 197 of DMP1.1. The unpurified rabbit anti-serum was used (1:5,000 dilution) for detection of unfused DMP1 proteins.
Membranes were incubated for 1.5 h with the appropriate antibody diluted according to manufacturer's instructions in TBST containing 6.5% non-fat dry milk. Membranes were washed three times for 5 min with TBST and, when required, incubated with the secondary antibody for another 1.5 h. Primary antibodies used were rabbit anti-GFP-HRP (Santa Cruz Biotechnology, Inc.), rabbit anti-DMP1 (Thermo Fisher Scientific Inc.), rabbit anti-BiP2 (Agrisera) for detection of ER-microsomes, rabbit anti-H+-ATPase (Agrisera) for detection of PM-derived vesicles and rabbit anti-v-ATPase (Agrisera) for detection of tonoplast-derived vesicles. Goat anti-mouse or anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Inc) were used as secondary antibodies. Custom anti-DMP1 primary antibodies were raised against the DMP1 C-terminal peptide KRSGIGYAPIAEEVGAE corresponding to aa 181 to 197 of DMP1.1. The unpurified rabbit anti-serum was used (1:5,000 dilution) for detection of unfused DMP1 proteins.
5
Western Blot Analysis of Lung Cancer Cells
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Lung cancer cell lysates were prepared using RIPA buffer (BestBio, Shanghai, China) and equalized for protein concentrations with a BCA Kit (Pierce, Rockford, IL) according to the manufacturers' recommendations. An equal amount of whole cell lysates were resolved using SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) followed by incubation with primary mouse monoclonal antibodies against human EZH2 (BD Biosciences), flag (Multisciences, Hangzhou, China), rabbit polyclonal antibody against human GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit monoclonal antibody against human NOTCH1 (Abcam, Shanghai, China) and goat anti-mouse or anti-rabbit IgG-HRP (Santa Cruz Biotechnology). The immunoreactive proteins were detected with SuperSignal® West Pico Chemiluminescent Substrate (Thermo, Rockford, IL) according to the manufacturer's instructions.
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