Cells were collected from the inner layer of the stomach from untreated (n = 2) and RAN + lime- treated mice for 300 days (n = 3). The cells were washed with ice-cold 0.1 M phosphate-buffered saline (PBS; pH 7.4), and total protein was extracted with lysis buffer containing 0.1% SDS, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 50 mM sodium fluoride, 100 U/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride. After centrifugation, the cell lysate was collected and the protein concentration was determined using the bicinchoninic acid protein assay. Equal amounts of protein (40 µg/well) were subjected to Novex Tris–Glycine 4–20% gradient gels, and electrophoresis was performed in a NuPAGE electrophoresis system (Invitrogen, California, USA). Then the proteins were transferred to a polyvinylidene difluoride membrane (Sigma) and probed with 1:1000 dilution of a mouse monoclonal antibody against securin (DCS-280; ab3305; Abcam, California, USA) and β-actin (AC-15; ab6276; Abcam, USA). Alkaline–phosphatase conjugated anti-mouse IgG (Abcam, USA) was used as the secondary antibody, and immunodetection was performed by treating the blot with the substrate solution of BCIP/NBT (Bangalore Genei, India).
Alkaline phosphatase conjugated anti mouse igg
Manufactured by Abcam
Sourced in United States
Alkaline–phosphatase conjugated anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG). The alkaline phosphatase enzyme conjugated to the antibody can be used to detect and quantify the presence of mouse IgG in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Nupage electrophoresis system, by Thermo Fisher Scientific (3 mentions)
Ab3305, by Abcam (3 mentions)
Ab6276, by Abcam (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Nbt bcip system, by Thermo Fisher Scientific (2 mentions)
Dcs 280, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
6 protocols using alkaline phosphatase conjugated anti mouse igg
1
Securin Expression in Stomach Cells
2
Quantitative Protein Expression Analysis
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The hearts were homogenized in lysis buffer containing (mmol/l) 150 NaCl, 50 Tris-HCl, 5 EDTA.2Na, and 1 MgCl2 plus protease inhibitor (Sigma Fast; Sigma, USA). The protein concentration was determined by the Lowry method, [22] (link) and bovine serum albumin (BSA) was used as the standard. Equal amounts of protein (50 µg) were separated by 10% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes incubated with mouse monoclonal antibodies for catalase (CAT; 1∶2000; Sigma, USA), rabbit polyclonal antibodies for superoxide dismutase (SOD-2; 1∶1000; Sigma, USA) and Gp91phox (1∶1000; BD, New Jersey, EUA) and rabbit polyclonal antibodies for AT1 (1∶500; Santa Cruz Biotechnology, CA, USA) and GAPDH (1∶1000; Santa Cruz Biotechnology, CA, USA). After washing, the membranes were incubated with either an alkaline phosphatase conjugated anti-mouse IgG (1∶3000, Abcam Inc., Cambridge, MA, USA) or an anti-rabbit antibody (1∶7000; Santa Cruz Biotechnology, CA, USA). The bands were visualized using a NBT/BCIP system (Invitrogen Corporation, CA, USA) and quantified using ImageJ software (National Institute of Health, NIH). The results were calculated using the ratio of the density of specific proteins to the corresponding GAPDH.
3
Analysis of Esophageal and Gastric Cells
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Cells were collected from the inner layer of esophagous and stomach from untreated (n = 5), RAN + lime and PRE + RAN + lime treated mice for 180 and 260 (n = 5 per point) days. In case of 180 days treatment with ECGU + RAN + lime 4 mice were used as a treated group. The cells were washed with ice-cold 0.1 M phosphate-buffered saline (PBS; pH 7.4) and total protein was extracted with a lysis buffer containing 0.1% SDS, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 50 mM sodium fluoride, 100 U/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride. After centrifugation, the cell lysate was collected and the protein concentration was determined using the bicinchoninic acid protein assay. Equal amount of protein (40 µg/well) were subjected to Novex Tris-Glycine 4–20% gradient gels and electrophoresis was performed in NuPAGE electrophoresis system (Invitrogen, USA). Then the proteins were transferred to a polyvinylidene difluoride membrane (Sigma) and probed with 1:1000 dilution of a mouse monoclonal antibody against p53 (PAb 240; ab-26; Abcam, USA), Securin (DCS-280; ab3305; Abcam, USA) and β-actin (AC-15; ab6276; Abcam, USA). Alkaline–phosphatase conjugated anti-mouse IgG (Abcam, USA) used as secondary antibodies and immunodetection was performed by treating the blot with the substrate solution of BCIP/NBT (Bangalore Genei, India).
4
Western Blot Analysis of Coronary Arteries
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The coronary arteries were pooled with frozen tissue from 3 rats (considered as n=1),
and the total number of pooled samples per group was n=4. The samples were
homogenized in lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 2 mM
Na, and 1 mM MgCl2 plus protease inhibitors (Sigma Fast, USA). The protein
concentration was determined by the Lowry method (22 (link)) and bovine serum albumin was used as the standard. Equal amounts of
protein were denatured and separated by 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore,
Germany). The membranes were blocked with 5% bovine serum albumin at room temperature
in TBS buffer plus Tween 20 (0.1%) before incubation with monoclonal anti-mouse for
eNOS (1:1500; BD Biosciences, USA), polyclonal anti-mouse for estrogen receptor alpha
(ER-α) (1:500; Santa Cruz Biotechnology, USA), and polyclonal anti-mouse for β-actin
(1:1500; Sigma). After washing, the membranes were incubated with an alkaline
phosphatase conjugated anti-mouse IgG (1:3000; Abcam Inc., USA). The bands were
visualized using an NBT/BCIP system (nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl-1-phosphate; Invitrogen, USA) and quantified
using ImageJ software (National Institutes of Health, USA).
and the total number of pooled samples per group was n=4. The samples were
homogenized in lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 2 mM
Na, and 1 mM MgCl2 plus protease inhibitors (Sigma Fast, USA). The protein
concentration was determined by the Lowry method (22 (link)) and bovine serum albumin was used as the standard. Equal amounts of
protein were denatured and separated by 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore,
Germany). The membranes were blocked with 5% bovine serum albumin at room temperature
in TBS buffer plus Tween 20 (0.1%) before incubation with monoclonal anti-mouse for
eNOS (1:1500; BD Biosciences, USA), polyclonal anti-mouse for estrogen receptor alpha
(ER-α) (1:500; Santa Cruz Biotechnology, USA), and polyclonal anti-mouse for β-actin
(1:1500; Sigma). After washing, the membranes were incubated with an alkaline
phosphatase conjugated anti-mouse IgG (1:3000; Abcam Inc., USA). The bands were
visualized using an NBT/BCIP system (nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl-1-phosphate; Invitrogen, USA) and quantified
using ImageJ software (National Institutes of Health, USA).
5
Oxidative Stress Protein Analysis in Coronary Samples
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Coronary samples were homogenized in a lysis buffer containing 150 mM NaCl, 50 mM
Tris-HCl, 5 mM Na2EDTA, and 1 mM MgCl2, plus a protease
inhibitor (Sigma Fast; Sigma). Protein concentration was determined by the Lowry
method (27 ) and bovine serum albumin (BSA) was
used as the standard. The same amount of proteins (50 µg) were denatured and
separated by SDS-PAGE (10%) and transferred onto a PVDF membrane (Millipore,
Germany). Membranes were blocked with 5% BSA at room temperature in a TBS buffer plus
Tween 20 (0.1%) before incubation with polyclonal anti-mouse antibody for
Mn-superoxide dismutase 2 (SOD-2; 1:1000-Sigma), monoclonal anti-mouse antibody for
catalase (1:2000-Sigma), and polyclonal anti-mouse antibody for β-actin
(1:1500-Sigma). After washing, the membranes were incubated with an alkaline
phosphatase conjugated anti-mouse IgG (1:3000, Abcam Inc., USA). The bands were
visualized using an NBT/BCIP system (Invitrogen Corporation, USA) and quantified
using ImageJ software (National Institute of Health, USA).
Tris-HCl, 5 mM Na2EDTA, and 1 mM MgCl2, plus a protease
inhibitor (Sigma Fast; Sigma). Protein concentration was determined by the Lowry
method (27 ) and bovine serum albumin (BSA) was
used as the standard. The same amount of proteins (50 µg) were denatured and
separated by SDS-PAGE (10%) and transferred onto a PVDF membrane (Millipore,
Germany). Membranes were blocked with 5% BSA at room temperature in a TBS buffer plus
Tween 20 (0.1%) before incubation with polyclonal anti-mouse antibody for
Mn-superoxide dismutase 2 (SOD-2; 1:1000-Sigma), monoclonal anti-mouse antibody for
catalase (1:2000-Sigma), and polyclonal anti-mouse antibody for β-actin
(1:1500-Sigma). After washing, the membranes were incubated with an alkaline
phosphatase conjugated anti-mouse IgG (1:3000, Abcam Inc., USA). The bands were
visualized using an NBT/BCIP system (Invitrogen Corporation, USA) and quantified
using ImageJ software (National Institute of Health, USA).
6
Securin Expression in Stomach Cells
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Cells were collected from the inner layer of stomach from untreated (n=2) and RAN+lime treated mice for 300 days (n=3). The cells were washed with ice-cold 0.1 M phosphate-buffered saline (PBS; pH 7.4) and total protein was extracted with a lysis buffer containing 0.1% SDS, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 50 mM sodium uoride, 100 U/ml aprotinin and 1 mM phenylmethylsulfonyl uoride. After centrifugation, the cell lysate was collected and the protein concentration was determined using the bicinchoninic acid protein assay. Equal amount of protein (40 µg/well) was subjected to Novex Tris-Glycine 4-20% gradient gels and electrophoresis was performed in NuPAGE electrophoresis system (Invitrogen, California, USA). Then the proteins were transferred to a polyvinylidene di uoride membrane (Sigma) and probed with 1:1000 dilution of a mouse monoclonal antibody against Securin (DCS-280; ab3305; Abcam, California, USA) and β-actin (AC-15; ab6276; Abcam, USA). Alkaline-phosphatase conjugated anti-mouse IgG (Abcam, USA) used as secondary antibodies and immunodetection was performed by treating the blot with the substrate solution of BCIP/NBT (Bangalore Genei, India).
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