Frozen ileum tissue (ca. 100 mg) was homogenized with cell lysis buffer (500 μl) (Cat EPX-99999-000 eBioscience, San Diego, USA) with 0.1 % proteinase inhibitor cocktail (Sigma, St. Louis, MO) and 1 % NP40 in ceramic bead tubes using Fast Prep-24™ 5G machine (M.P. Biomedical LLC, California, USA) at speed 6.0 msec for 30 s (twice), and then centrifuged at 12,000 g for 15 min at 4 °C. Supernatants were collected and 100 μg of protein was used for Western blot analyses. Membranes were probed with 1:1000 dilution of rabbit anti-VE-cadherin, rabbit anti-occludin, and rabbit anti-claudin 3 (Abcam, Cambridge, MA) primary antibodies and subsequently incubated with 1:10,000 dilution of goat-anti-rabbit HRP (BioRad Laboratories Inc., California). Detection was performed using a chemilumiescence system (super signal west chemiluminescent substrate, Thermo Scientific). Immuno-quantitation was performed by densitometric scanning of the blot and normalized against the signal from β-actin (Sigma Aldrich, St Louis, MO) using Image Quant software (Image Quant TL 8.1 Version). We measured calcium levels in urine from breast-fed and formula fed piglets using colorimetric assay from Bio Vision (Catalog#K380-250) as per manufacturer’s instructions.
Supersignal west chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
SuperSignal West chemiluminescent substrate is a reagent used in western blotting applications to detect and quantify proteins. It produces a luminescent signal when interacting with horseradish peroxidase-labeled detection antibodies, allowing for sensitive and specific protein visualization.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Gradient tris glycine sds polyacrylamide gels, by Thermo Fisher Scientific (3 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Proteinase inhibitor cocktail, by Merck Group (2 mentions)
Goat anti rabbit hrp, by Bio-Rad (2 mentions)
Anti 5fc antiserum, by Active Motif (2 mentions)
Hrp conjugated anti rabbit igg secondary antibody, by CWBIO (2 mentions)
Amersham hybond n membrane, by GE Healthcare (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Goat anti rabbit igg hrp sc 2004, by Santa Cruz Biotechnology (2 mentions)
Anti myb, by Merck Group (2 mentions)
Rabbit anti occludin, by Abcam (1 mentions)
Fast prep 24 5g machine, by MP Biomedicals (1 mentions)
Rabbit anti claudin 3, by Abcam (1 mentions)
Rabbit anti ve cadherin, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Anti ve cadherin, by Abcam (1 mentions)
27 protocols using supersignal west chemiluminescent substrate
1
Quantitative Analysis of Intestinal Tight Junctions
2
Proteomic analysis of colon tissue
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Frozen colon tissue (~100 mg) was homogenized in cell lysis buffer (500 μl) (Cat EPX-99999-000 eBioscience, San Diego, USA) containing 0.1% proteinase inhibitor cocktail (Sigma, St. Louis, MO) and 1% NP40, in ceramic bead tubes using Fast Prep-24TM 5G machine (M.P. Biomedical LLC, California, USA) at a speed 6.0 ms for 30 s (twice). Samples were centrifuged at 12,000 g for 15 min at 4 °C, supernatants were collected and protein concentration was determined using Bio-Rad protein estimation kit (BioRad). 100 μg of protein was subjected to Western Blot analyses using 8% acrylamide gel. Membranes were probed with anti-pig primary antibodies raised in rabbit overnight at 4 °C and subsequently incubated with 1:10,000 dilution of goat-anti-rabbit HRP for 1 h at room temperature (BioRad Laboratories Inc., California). We used 1:1000 dilution of anti-VE-cadherin, anti-catenin, anti-HSP 27 (Abcam, Cambridge, MA) as primary antibodies. Detection was performed using a chemiluminescence system (super signal west chemiluminescent substrate, Thermo Scientific). Image Quant software (Image Quant TL 8.1 Version) was used for densitometric analysis. Anti-rabbit vinculin (Abcam ab73412) that cross reacts with pigs was used as a housekeeping protein for normalization of a target protein.
3
Quantitative 5fC Detection in DNA
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Chemical labeling of 76mer model DNA containing single 5fC or Tdg−/− mESC gDNA was performed as described above. For the dot blot assay, different amounts of model DNA or gDNA were denatured in advance with NaOH solution (final concentration of 0.15M), spotted on the Amersham Hybond-N+ membrane (GE Healthcare) and air-dried for 5 min. The membrane was UV crosslinked and then blocked with 5% nonfat milk in 1x TBST at room temperature for 2 h. The membrane was then incubated with anti-5fC antiserum (Active Motif, 61223, 1:2,500 dilution) overnight at 4 °C followed by 1× TBST washing three times. After incubation with HRP-conjugated anti-rabbit IgG secondary antibody (CW Biotech, CW0103, 1:10,00 dilution) at room temperature for 1 h and washing with 1× TBST for three times, the membrane was supplied with 1 mL SuperSignal West Chemiluminescent Substrate (Thermo Scientific) and then visualized by chemiluminescence exposure.
4
Immunoblotting of Cellular Signaling Proteins
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Cells were lysed in cold RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentration was determined by BCA assay (Thermo Fischer Scientific). Equal amounts of protein were resolved by 4-10% Bis-Tris/PAGE, transferred to PVDF membranes (BioRad) and probed overnight at 4°C with the following primary antibodies: anti-Clusterin-α (1:3000), anti-Sox2 (1:2000), anti-HSP90 (1:2000), anti-Cleaved PARP (1:2000), anti-pSer807/Ser811-Rb (1:2000), anti-AKT (1:2000), anti-CDK4 (1:2000), anti-HER2 (1:2000), anti-c-Raf (1:2000), anti-EGFR (1:2000), anti-IGF-1R (1:2000), anti-YAP (1:2000), anti-flag (1:2000), anti-β-actin (1:20000). Secondary antibodies were anti-goat-HRP (Santa Cruz sc2020; 1:5000), anti-mouse-HRP (Cell Signaling 7076; 1:5000) or anti-rabbit-HRP (Cell Signaling 7074; 1:5000). Blots were developed by using Immobilon Western Chemiluminescent HRP substrate (Millipore) or SuperSignal West Chemiluminescent substrate (Thermo Fisher Scientific), and imaged in ChemiDoc MP imaging system (BioRad).
5
Immunoblotting Procedure for Protein Analysis
6
Western Blotting Experimental Workflow
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Western blotting was performed as described (21 (link), 22 (link)). Cultured cells were washed with ice-cold PBS and lysed with RIPA lysis buffer (R0278; Sigma-Aldrich) containing protease and phosphatase inhibitor cocktails (P8340, P5726, and P0044; Sigma-Aldrich). Protein concentrations were determined using a BCA Protein Assay Kit (23227, Thermo Fisher Scientific). All samples were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. For blocking, 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) or 5% bovine serum albumin (BSA) in TBS-T was used for non-phosphorylated and phosphorylated proteins, respectively. After blocking, the membranes were incubated with the indicated primary antibodies, followed by incubation with appropriate horseradish peroxidase-conjugated species-specific secondary antibodies. Bands were detected using the SuperSignal West chemiluminescent substrate (Thermo Fisher Scientific) and visualized using ImageQuant LAS-4000 (GE Healthcare, Little Chalfont, UK). Actin was used as the loading control to normalize the amount of protein.
7
Western Blot Protein Detection
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Cells were lysed with 2x SDS sample buffer (100 mM Tris–HCl, pH 6.8, 5 mM EDTA/Na, 4% SDS, 10% glycerol, 0.4 M DTT, 0.02% bromophenol blue). After SDS-PAGE proteins were blotted. After blocking, membranes were incubated overnight with the primary antibodies and, after washing, with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA). For loading controls membranes were stripped in acidic buffer (0.2 M glycine, 0.1% SDS, 1% Tween-20, pH 2.2) and re-probed. Proteins were revealed on auto-radiographic films (GE Healthcare, Piscataway, NJ, USA) by Super Signal West Chemiluminescent Substrate (ThermoFisher Scientific, Waltham, MA, USA) and quantified using ImageJ64 .
8
Western Blot Analysis of Protein Samples
9
Western Blot Analysis of Protein Samples
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NHEKs were washed with ice-cold phosphate-buffered saline and lysed with a radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Protein samples (8 μg/lane) were resolved by SDS–PAGE and transferred to polyvinylidene fluoride membranes. After blocking with 5% BSA in Tris-buffered saline with Tween 20, the membranes were incubated with primary antibodies, followed by further incubation with the appropriate horseradish peroxidase-conjugated species-specific secondary antibodies. All primary antibodies were diluted at a ratio of 1:1000, and secondary antibodies were diluted at 1:10,000. Bands were detected using a SuperSignal West chemiluminescent substrate (Thermo Fisher Scientific) and visualized using ImageQuant LAS-4000 (GE Healthcare, Little Chalfont, UK). Actin was used as a loading control to normalize the amount of protein. The intensities of the bands were quantified using ImageJ.
10
Quantitative 5fC Detection in DNA
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