RIPA buffer (Solarbio, Shanghai, China) was used to lyse cells and a BCA kit (Beyotime, China) was used to quantify protein levels. β-Actin (AbMART, Shanghai, China) was used as a loading control. The primary antibody specific for Timm13 was obtained from NOVUS (USA; NBP2-13431). Anti-rabbit and anti-mouse secondary antibodies were obtained from Invitrogen (Carlsbad, USA). An Odyssey two-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA) was employed to scan the blots. The quantitative analysis of grey values was performed using ImageJ software (NIH, USA).
Anti rabbit and anti mouse secondary antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-rabbit and anti-mouse secondary antibodies are laboratory reagents used to detect the presence of primary antibodies that have been produced in rabbits or mice. These secondary antibodies are designed to bind to the Fc region of the primary antibodies, allowing for their visualization or detection in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey two color infrared laser imaging system, by LI COR (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
β actin, by Abmart (1 mentions)
Labworks image acquisition and analysis software, by Analytik Jena (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Pten a2b1, by Santa Cruz Biotechnology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Genistein, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by GenDEPOT (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti p iκbα, by Cell Signaling Technology (1 mentions)
Anti actin, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Lys40, by Cell Signaling Technology (1 mentions)
5 protocols using anti rabbit and anti mouse secondary antibodies
1
Quantifying Timm13 Protein Levels via Western Blot
2
PTEN and AKT Protein Analysis
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Protein extraction was performed using a lysis buffer containing a protease inhibitor cocktail (Complete; Sigma-Aldrich), and protein quantification was performed using the bicinchoninic acid method (Pierce; Rockford, IL). 50 μg of protein per condition was run on SDS-PAGE gels and then transferred onto PVDF membranes (BioRad), which were subsequently blocked for 1 h with a solution of 5% skim milk/in TBS/Tween 0.1%. Membranes were incubated overnight with primary antibodies against PTEN (A2B1) (diluted 1:250; Cat No. 7974, Santa Cruz Biotechnology), AKT (diluted 1:5000; Cat No. 4691, Cell Signaling Technology), phospho-AKT (diluted 1:1000; Cat No. 9271, Cell Signaling Technology), β-Actin (diluted 1:10,000; Cat No. A3854, Sigma Aldrich), ERK1/2 (diluted 1:5000; Cat No. 9102S, Cell Signaling Technology) and pERK1/2 (diluted 1:500; Cat No. 9106S, Cell Signaling Technology). Subsequent washes were performed with TBS/Tween 0.1% and then membranes were incubated for 1 h with anti-rabbit and anti-mouse secondary antibodies diluted 1:3000 (Invitrogen), followed by washes with TBS/0.1% Tween. The presence of the proteins was revealed by chemiluminescence (Perkin-Elmer). Images were captured and analyzed using a UVP BioImaging System and LabWorks Image Acquisition and Analysis Software (UVP, Inc. Upland, CA).
3
Genistein Modulates Breast Cancer Pathways
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Genistein was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s Medium (DMEM) and Rosewell Park Memorial Institute (RPMI) 1640 medium were obtained from Gibco BRL (Grand Island, NY, USA). FBS was supplied from GenDEPOT (Barker, TX, USA). TRIzol®, SYBR® safe DNA gel stain and Lipofectamine® RNAiMAX were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against BRCA1, P-Akt and Akt were obtained from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against cyclin B1 and actin were products of Santa Cruz Biotechnology (Santa Cruz, CA, USA). GPR30 and Nrf2 primary antibodies were supplied from Abcam (Cambridge, MA, USA). Anti-rabbit and anti-mouse secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA).
4
Immunostaining Protocol for Cell Signaling
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This study used Anti-p-IκBα (Cell Signaling Technology, Danvers, MA, USA), Anti-Actin (Cell Signaling Technology, Danvers, MA, USA), Anti-p65 (Cell signaling Technology, Danvers, MA, USA), Anti-Vimentin (Cell Signaling Technology, Danvers, MA, USA) and Ki-67 (Cell Signaling Technology, Danvers, MA, USA) and secondary anti-rabbit and anti-mouse antibodies (Invitrogen, Carlsbad, CA, USA). Anti-ATX was gifted by Dr. T. Clair (National Cancer Institute, Bethesda, MA, USA), and IOA-289 was a gift from iOnctura (Genève, Switzerland).
5
Visualizing Acetylated Microtubules in Breast Cancer Cells
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Immunochemistry was performed on BoM-MDA-231 control and Runx2 knockdown as previously described [2 (link),17 (link)]. To visualize acetylated microtubules, cells were seeded in 8-well glass chamber slides and incubated overnight. Cells were subjected to control, nocodazole, or vinblastine treatment followed by fixation and imaging. Primary antibodies for α-tubulin and acetylated α-tubulin (Lys40) were obtained from Cell Signaling Technologies (CST) and used at 1:1000 dilutions. Secondary anti-rabbit and anti-mouse antibodies were obtained from Invitrogen and used at 1:1000 dilutions. Anti-rabbit secondary antibody was conjugated with AF-594. Slides were mounted with prolonged antifade reagent with DAPI (Invitrogen, Waltham, MA, USA). Cells were imaged using the Zeiss (LSM70) Confocal microscope equipped with a 63X oil immersion lens. Images were quantified using ImageJ software. Quantification provides integrated density reflecting the intensity of the stain within the region. The data shown provide the mean, and error bars represent the SD.
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