Total protein was extracted by sonication of liver homogenates for determinations of cytokines. Protein levels were determined by the Lowry method (Waterborg, 2009 ). Equal amounts of protein samples were separated in 10% and 12% polyacrylamide gels and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk for 3 h at 4°C. The membranes were incubated with one of the following primary antibodies: β-actin (sc-1615), TNFα (sc-12744). These antibodies were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, United States), and diluted to 1:2000. Antibody for IL-6 (ab6672) was obtained from Abcam (Cambridge, United Kingdom) and diluted to 1:4000. After three washes with TBS-T, the membranes were incubated at 4°C for 3 h with 1:2000 donkey anti-rabbit IgG-HRP (sc-2305) and m-IgGκ BP-HRP (sc-516102), obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, United States). Finally, the images were obtained in a multifunctional gel imaging system (Bio-Rad Laboratories, Hercules, CA, United States). The densities of protein bands were quantified using the ImageJ software (Schneider et al., 2012 (link)).
M iggκ bp hrp sc 516102
Manufactured by Santa Cruz Biotechnology
Sourced in United States
M-IgGκ BP-HRP (sc-516102) is a horseradish peroxidase (HRP)-conjugated secondary antibody that binds to the kappa light chain of mouse immunoglobulin G (IgG). It is designed for use in various immunoassay techniques, such as Western blotting and immunohistochemistry, to detect and visualize mouse kappa light chain-containing proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti rabbit igg hrp sc 2357, by Santa Cruz Biotechnology (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Isopropanol, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Multifunctional gel imaging system, by Bio-Rad (1 mentions)
Sample buffer, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Chemiluminescent reagent, by Cytiva (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Tbs t, by Thermo Fisher Scientific (1 mentions)
Bradford method, by Merck Group (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti β actin igg, by Merck Group (1 mentions)
A0545, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Anti gapdh g9545, by Merck Group (1 mentions)
Ecl western blotting detection system, by Cytiva (1 mentions)
Hybond ecl, by Cytiva (1 mentions)
22 protocols using m iggκ bp hrp sc 516102
1
Quantitative Protein Expression Analysis
2
Quantifying Protein Levels via Western Blot
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Western blotting was performed to measure protein changes. In brief, the samples (tissue and cell samples) were lysed in RIPA buffer (P0013C, Beyotime, Guangzhou, China) and mixed with sample buffer (No. 1610737, Bio–Rad) at a 1:1 ratio. Equal amounts of protein samples were separated by 10% SDS–PAGE and blotted onto PVDF membranes after transmembrane electrophoresis. The antibodies used included Runx1 (No. 39000, ChIP grade, Active motif, Carlsbad, CA) and FLAG (ab125243, Abcam, Cambridge, UK and 9A3, Cell Signaling Technology, Boston, MA, 1:1 000 dilution). The secondary antibodies included mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz Biotech, Delaware Avenue, CA, 1:2 000 dilution) and m-IgGκ BP-HRP (sc-516102, Santa Cruz Biotech, 1:2 000 dilution).
3
Western Blot Analysis of LGR5 Protein
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RIPA (Radio-Immunoprecipitation Assay) lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract proteins from cells, and protein concentration was established by Bradford method (Sigma–Aldrich, Madrid, Spain). Separation of proteins was carried out in 8% SDS-PAGE gels in which 40 µg of proteins of each sample were loaded previously heated at 65 °C for 20 minutes. Then, proteins were electrotransferred to a nitrocellulose membrane (45 µm pore size) (Millipore, Burlington, MA, USA) and blocked for 1 hour in 5% milk in TBS with 0.1% Tween-20 (TBS-T) (Bio-Rad, Hercules, CA, USA). After several washes in TBS-T, the incubation of the anti-LGR5 primary antibody (1:750) (Anti-LGR5 (OTI2A2): MA5-25,644; Invitrogen, Waltham, Massachusetts, USA) was performed overnight at 4 °C. Then, a secondary antibody (m-IgGκBP-HRP: sc-516,102; Santa Cruz Biotechnology, Dallas, Texas, EEUU) was added (1:2000) for 1 hour at room temperature. Chemiluminescent reagents (Amersham Biosciences, Saint Louis, MO, USA) were used for the detection of membrane-bound antibodies. Besides, anti-β-actin IgG (A3854, Sigma Aldrich, Madrid, Spain) (1:25,000 dilution) was used as an endogenous control. The open-source Fiji image analysis software was used to quantify the bands obtained to calculate relative protein expression.28 (link)
4
Immunoblotting of mCherry and GFP Proteins
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Protein extraction and immunoblotting were performed as described in Zhang et al. (2018) (link). Primary antibodies were used at the following dilutions: anti-mCherry (ab183628; Abcam), 1:3,000; anti-GFP from mouse IgG1κ (clones 7.1 and 13.1; Roche), 1:1,000. The secondary antibodies used were anti-rabbit horseradish peroxidase conjugate (A0545; Sigma) diluted 1:50,000 for mCherry or m-IgGκ BP-HRP (sc-516102; Santa Cruz Biotechnology) diluted 1:5,000 for GFP. Blots were then washed and developed with enhanced chemiluminescence reagent SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), as required.
5
Western Blotting Technique for Protein Analysis
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5 μg of protein per lane was separated on 10% SDS/PAGE gel and transferred onto a Hybond ECL® (enhanced chemiluminescence) membrane (Amersham Biosciences). After blocking with 5% milk for 1 h, the membrane was incubated overnight at 4°C with primary antibodies diluted 1:1000 followed by a further 1 h incubation with the corresponding horseradish peroxidase-conjugated secondary antibody (Bio-Rad) at a 1:10000 dilution, and the signal was detected with the ECL® Western blotting detection system (Amersham Biosciences). The following primary antibodies were used for immunoblotting: Anti-AKAP1 (D9C5), Anti-COX4 (3E11), Anti-Mios (D12C6), Anti-Sestrin2 (D186) were from Cell Signaling Technology; Anti-Actin (ab3280) was from Abcam; Anti-GAPDH (G9545) was from Sigma-Aldrich. Secondary antibodies were from Santa Cruz Biotechnology: m-IgGκ BP-HRP (sc-516102) and mouse anti-rabbit IgG-HRP (sc-2357).
6
Western Blot Analysis of GAL4 DBD:BRCA1 Fusion
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The transfection experiments described under Transfection and cell cultivation were scaled to a 12-well plate setup for expression analysis of the GAL4 DBD:BRCA1(aa 1396–1863) fusion protein constructs by western blot analysis. Ten microgramme of sample protein was run on a 10% Mini-PROTEAN® TGX™ gel (Bio-Rad Laboratories, Hercules, CA, USA) and blotted onto 0.2 μm Nitrocellulose Membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% BSA. Primary immunoblot staining was done using BRCA1 (D-9):sc-6954 (Santa Cruz Biotechnology, Dallas, TX, USA) in a 1:1000 dilution, overnight at 4 °C, and followed by staining with secondary antibody m-IgGκ BP-HRP: sc-516102 (Santa Cruz Biotechnology, Dallas, TX, USA) (1:1000) for 1 h at room temperature, before exposure using the ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) with ImageQuant™ LAS 4000 and ImageQuant™ TL 1D v8.1 (GE Healthcare, Buckinghamshire, UK).
7
Western Blot Analysis of Mitophagy Markers
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Cell lysates were prepared in RIPA buffer plus proteinase inhibitors. Samples containing (20 µg) were separated by 10–15% SDS-PAGE, transferred to nitrocellulose membranes, blocked in 5% nonfat milk for 1h at RT and incubated overnight at 4 °C with the primary antibodies: MFN2 (H-68, Santa Cruz, sc-50331, dilution 1:1000, rabbit); Optineurin (C-2, Santa Cruz, sc-166576, dilution 1:1000, mouse); PINK1 (BC100-494, Novus Biologicals, dilution 1:2000, rabbit); Parkin (PRK8, ab77924, Abcam, dilution 1:2000, mouse); β-Actin (Sigma-Aldrich, A3854, dilution 1:40,000 conjugated to HRP). Secondary antibody incubation was performed for 1 h at RT using anti-mouse (m-IgGκ BP-HRP sc-516102, Santa Cruz, 1:10,000) and anti-rabbit (goat anti-rabbit IgG-HRP sc-2054, Santa Cruz, 1:10,000). Proteins were detected using ECL western blotting substrate (Pierce, Waltham, MA, USA), Clarity and Clarity Max (Bio-Rad, Hercules, CA, USA) and ChemiDoc Imaging System (Bio-Rad).
8
Investigating Gastric Cancer Cell Lines
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Human gastric cancer cell lines SGC-7901 and BGC-823 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C in a humidified incubator with an atmosphere of 5% CO2. N-acetyl-L-cysteine (NAC), L-glutathione (GSH) and cisplatin were purchased from Sigma (St. Louis, MO, USA). BMS-582949 and SP600125 were purchased from Selleck Chemicals (Houston, TX, USA). WZ35 was synthesized in our laboratory as previously described [13 (link)]. Antibodies including anti-Bcl-2 (sc-7382, 1:50), anti-TrxR1 (sc-28,321, 1:200), anti-GAPDH (sc-47,724, 1:200), mouse anti-rabbit IgG-HRP (sc-2357, 1:2000) and m-IgGκ BP-HRP (sc-516,102, 1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies including anti-p-p38 (4631, 1:1000), anti-p38 (9212, 1:1000), anti-p-JNK (4668, 1:1000) and anti-JNK (9252, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-Ki-67 (ab15580, 1:1000) antibody was purchased from Abcam (Cambridge, MA, USA). FITC Annexin V apoptosis Detection Kit I and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA).
9
Hepatocyte-specific G3PP Deletion Validation
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After 4 weeks following AAV8 viral infection, mice were anesthetized and the livers were perfused with collagenase buffer for hepatocyte preparation (see below for details) and visceral adipose tissue, brain and skeletal muscle were collected. Mouse tissues and isolated hepatocytes were lysed in RIPA buffer (10 mM Tris–HCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate and 140 mM NaCl) and the protein extracts were used for western blot analysis to validate hepatocyte-specific G3PP deletion (G3PP primary antibody: PGP Antibody (E−10) (sc-390 883), secondary antibody: m-IgGκ BP-HRP (sc-516 102) both from Santa Cruz Biotechnology). For all tissues, α-tubulin was used as the gel loading control.
10
Protein Expression Analysis in Cardiac Tissue
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Left ventricular myocardial biopsies or cells were lysed in RIPA protein lysis buffer containing PMSF and protease inhibition cocktail (Roche Diagnostics GmbH, Mannheim, Germany) using the Bead Ruptor 4 Mini Homogenizer (Omni International). Protein concentration was determined with a BCA protein assay kit (Thermo Scientific, Rockford, USA). Protein extracts were separated on a polyacrylamide gel and subsequently transferred to a PVDF membrane. The membrane was blocked in 5% non‐fat dry milk/TBS‐T buffer followed by overnight incubation at 4°C with primary antibody. After rinsing, the membrane was incubated with HRP conjugated secondary antibody and detected by use of the ECL detection kit (Bio‐Rad). Densitometric quantification of protein bands was performed with the Chemidoc Touch Imaging System (Bio‐Rad). Following antibodies were used: anti‐SERPINA3 rabbit monoclonal antibody (ab205197, Abcam, Cambridge), Akt antibody (#9272) (Cell Signaling Technologies, Massachusetts), and HRP conjugated goat anti‐rabbit IgG secondary antibody (TA1300233, Origene). GAPDH was used to normalize for different protein content using GAPDH antibody (sc‐47724) and mIgG κ BP‐HRP (sc‐516102) (Santa Cruz Biotechnology, Inc, Dallas, Texas).
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