Protein was extracted from SW480 and Lovo cells using RIPA lysis buffer (Beyotime, Shanghai, China). Briefly, the BCA (Beyotime, Shanghai, China) method was used to determine the protein density. Total protein (30 μg/sample) was separated using SDS-PAGE electrophoresis, then transferred to a PVDF membrane. Following blocking with 5% skimmed milk, the membrane was exposed to primary antibodies against CD44, EGFR, or β-catenin (all from ABCAm, Cambridge, MA, USA) for overnight at 4°C, then washed and exposed to the second antibody, goat anti-mouse IgG-HRP (Bioworld) for one hour at room temperature. The 350 μL ECL luminous substrate was added and images were formed in the darkroom. Antibodies to β-actin and GAPDH-HRP (Beyotime, Shanghai, China) were used as internal controls for the experiment.
Goat anti mouse igg hrp
Manufactured by Bioworld Technology
Sourced in United States
Goat anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to mouse immunoglobulin G (IgG) in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg hrp, by Bioworld Technology (3 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Tauroursodeoxycholic acid, by Merck Group (1 mentions)
Eif2α, by Bioworld Technology (1 mentions)
96 well plate, by Corning (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Elisa plate reader, by Bio-Rad (1 mentions)
Rnaiso, by Takara Bio (1 mentions)
Acrylamide bis solution, by Bio-Rad (1 mentions)
β actin antibody, by Bioworld Technology (1 mentions)
Vegf antibody, by Abcam (1 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Cyclin e, by ZenBio (1 mentions)
Myhc iib, by ABclonal (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Cyclin d1, by ZenBio (1 mentions)
6 protocols using goat anti mouse igg hrp
1
Protein Expression Analysis in Colon Cancer
2
Investigating the UPR Pathway Components
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The primary antibodies used in the study were specific for GRP78 (Santa Cruz Biotechnology, sc-13968), GRP94 (Novus, NBP2-44690-0.02mg), eIF2α (phospho-S51) (Bioworld, BS4787), eIF2α (Bioworld, BS3651), IRE1 (phosphor S724) (Abcam, ab48187), IRE1 (Bioss, bs-8680R), ATF6 (ABclonal, A0202), ATF4 (ImmunoWay, YT1102), CHOP (Santa Cruz Biotechnology, sc-166682), CSFV-E2 (JBT, 2229), CSFV-Npro (a gift from Hunan agricultural university professor YuXingLong), Tubulin (Beyotime, AT819), and Actin (Beyotime, AT0003). The secondary antibodies Goat anti-Mouse IgG-HRP (Bioworld, BS12478) and Goat anti-Rabbit IgG-HRP (Bioworld, BS13278) were purchased from Bioworld Technology. Thapsigargin (Calbiochem®, 67526-95-8), 4μ8c (selleck.cn, S7272) and BIX (Sigma, SML1073) were dissolved in dimethyl sulfoxide (DMSO) at the indicated concentrations. Tauroursodeoxycholic acid (sodium salt, Merck-Millipore, 580549-1GM) and NH4CL (Damao Chemical Reagent, 12125-02-9) were dissolved in ddH2O at the indicated concentrations. Fluo4-am (BestBio, BB-48113) was diluted 50-folds with Hank’s Balanced Salt Solution (HBSS). pEGFP-N1 and pEGFP-GRP78 were prepared in our laboratory. Additionally, shRNA vector targeting the coding sequences of GRP78 and the scrambled shRNA were designed by and purchased from Cyagen.
3
ELISA Assessment of Humoral Antibody Titers
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ELISA was used to assess the humoral antibody titers in serum samples. The coated antigen protein was purchased from Sino Biological (Cat: 40592-V08H). RBD protein (2 μg/mL, 100 μL/well) was coated on 96-well plates (Corning) at 4 °C overnight and then blocked with 1% bovine serum albumin (Sigma) at 37 °C for 2 h. We then serially diluted the serum 2-fold, starting at 1:200, and added it to the plates. The plates were first incubated for 1 h at 37 °C, then washed three times with PBST (PBS containing 0.05% Tween-20), and then incubated for 1 h at 37 °C with goat anti-mouse IgG-HRP at a dilution of 1:25,000 (Cat: BS12478, Bioworld). We also tested for IgG subtypes, including IgG1, IgG2a, IgG2b, IgG2c, and IgG3 at a dilution of 1:5000 (Cat: 1071–05, 1081–05, 1091–05, 1078–05, 1101–05, Southern Biotech) by serially diluting the sera 2-fold from 1:200 and adding them to the plates the same as IgG. Then the plates were washed four times with PBST, and TMB substrate was used to produce the signals (Sigma). The colorimetric reaction was halted by applying a stop buffer, and the optical density (OD) was determined at 450 nm using an ELISA plate reader (Bio-Rad).
4
Confirming HAdV-3 Hexon Gene Expression
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The transcription of HAdV-3 hexon gene of rAd3H was confirmed by RT-PCR analysis as follows: Total RNA was extracted by RNAiso (TAKARA; Tokyo, Japan) and reverse transcribed into cDNA using the primer HVRR (5′-TTTCTGAAGTTCCACTCGTAGGTGTA-3′). This resultant cDNA was used as the template in a PCR amplification with the primer pairs, HVRF1 (5′-CAGGATGCTTCGGAGTACCTGAG-3′) and HVRR (Zhang et al. 2020 (link)). The PCR product was identified using agarose gel electrophoresis and DL10000 markers. Subsequent Western blot analysis confirmed expression of the hexon protein. A culture of rAd3H was frozen and thawed three times, centrifuged at 1000 ×g for 10 min, and the supernatant was collected. The samples were added to loading buffer, boiled for 10 min, electrophoresed in 5% concentrated gel, and 10% Tris-Tricine SDS-PAGE. After PAGE, the proteins were transferred onto nitrocellulose membrane by electrophoresis at 25 V for 40 min. The membrane was probed first by binding with mouse anti-HAdV-3-hexon antibody (Guangzhou HuYanSuo Medical Technology; Guangzhou, China) as the epitope-specific antibody (1:1000 dilution), and followed with goat anti-mouse IgG-HRP (Bioworld; Georgia, USA) as the secondary amplifying antibody. Luminol and hydrogen peroxide were used as chromogenic substrates to score the fluorescence signal.
5
Rat Model of Erythropoietin Signaling
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Human erythropoietin (10,000 U, Shenyang Sunshine Pharmaceutical Co., Ltd. Shenyang, China); Goat polyclonal to PECAM-1 Antibody (1:50, Santa Cruz, CA, USA); Rabbit anti-sheep SP immunohistochemical staining kit and citrate antigen retrieval solution (pH 6.0) (Fuzhou Maixin Biotech. Co., Ltd., Fuzhou, China); Hematoxylin (Sigma, St. Louis, USA); PVDF membranes (Millipore Company, MA, USA); Polylysine solution (0.1%) and DAB Horseradish Peroxidase Color Development Kit (Beijing Golden Bridge Biotechnology Co., Ltd., Beijing, China); Prestained Color Protein Molecular Weight Marker (Fermentas, Waltham, MA , USA); Tris-Hcl/SDS solutions (1.5 mM, pH 8.8; and 0.5 mM, pH 6.8, Sangon Biotechnology, Shanghai, China); Acrylamide/Bis solution (30%, Bio-Rad, California, USA); BeyoECL Plus kit, SDS-PAGE Sample Loading Buffer (5×), Western blot, IP cell lysates, PMSF together with BCA protein assay kit (Beyotime Biotechnology, Shanghai, China); Skim milk powder (Yili Group, Inner Mongolia, China); VEGF Antibody (Abcam, Cambridge Science Park, UK); β-actin Antibody, goat anti-Rabbit IgG-HRP, and goat anti-Mouse IgG-HRP (Bioworld, Minnesota, USA).
Eighteen SD male rats and eighteen SD female rats were supplied by Hangzhou hi-biotechnology Co., Ltd. (Hangzhou, China).
Eighteen SD male rats and eighteen SD female rats were supplied by Hangzhou hi-biotechnology Co., Ltd. (Hangzhou, China).
6
Protein Expression Analysis in Cell Lines
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The cells were lysed using RIPA lysis buffer (Solarbio Life Sciences, Beijing, China) to obtain proteins, which were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a 0.45-mm polyvinylidene uoride membrane (Sigma, St. Louis, MO), and sealed with 5% skim milk for 2 h at room temperature. The cells transfected for 48 h were incubated overnight at 4°C with the following primary antibodies: PCNA, Cyclin D1, Cyclin E, and Tubulin (ZenBio, Chengdu, China).
Further, the cells transfected and differentiated for 96 h were incubated overnight with following primary antibodies: MYOG, MyHC, Tubulin (ZenBio), MyHC I, MyHC IIb, and GAPDH (ABclonal, Wuhan, China).
After washing with Tris-buffered saline with Tween 20, the secondary antibody goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP (Bioworld, Minneapolis, MN) was added, followed by incubation at room temperature for 1 h. Finally, enhanced chemiluminescence luminous uid (Solarbio Life Sciences) was used for band visualization.
Further, the cells transfected and differentiated for 96 h were incubated overnight with following primary antibodies: MYOG, MyHC, Tubulin (ZenBio), MyHC I, MyHC IIb, and GAPDH (ABclonal, Wuhan, China).
After washing with Tris-buffered saline with Tween 20, the secondary antibody goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP (Bioworld, Minneapolis, MN) was added, followed by incubation at room temperature for 1 h. Finally, enhanced chemiluminescence luminous uid (Solarbio Life Sciences) was used for band visualization.
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