Western blotting was used to measure the protein expression in HK-2 cells or mouse kidney tissue after treatment (Part of the the blots were cut prior to hybridisation with antibodies during blotting). Antibody binding was detected using a western blotting detection kit (Thermo Fisher Scientific). Captured western blotting bands using an ultrasensitive multifunction imager (AI600RGB; General Electric, Boston, MA, USA). Bands were quantified with ImageJ and normalized to GAPDH expression.
Western blotting detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Western blotting detection kit is a comprehensive set of reagents designed for the detection and analysis of specific proteins in a complex sample. The kit includes all the necessary components for the visualization and quantification of target proteins using the Western blotting technique.
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Lab products found in correlation
Ai600rgb, by GE Healthcare (1 mentions)
Scion image software, by Techcomp Instruments (1 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Abcam (1 mentions)
Rabbit anti bcl 2 antibody, by Abcam (1 mentions)
Rabbit anti caspase 3 antibody, by Abcam (1 mentions)
Rabbit anti bax antibody, by Abcam (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Phosphor erk1 2, by Cell Signaling Technology (1 mentions)
4 protocols using western blotting detection kit
1
Protein Expression Quantification in Cells
2
Aβ-Induced Signaling in HeLa Cells
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HeLa cells transfected with 0.5 μg of empty vector, CALHM1, or P86L‐CALHM1 and treated or not with 5 μm of a mixture of protofibrils and oligomers of Aβ1–42 for 24 h were lysed with 100 μL of cold lysis buffer containing 1% Nonidet P‐40, 10% glycerol, 137 mm NaCl, 20 mm Tris‐HCl pH 7.5, 1 mg mL−1 leupeptin, 1 mm phenylmethylsulfonyl fluoride, 20 mm NaF, 1 mm Na4P2O7, and 1 mm Na3PO4. Once the amount of protein was quantified using the BCA Protein Assay Kit reagent, electrophoresis was performed by running 30 μg of protein in polyacrylamide gels for 2 h at constant amperage. Proteins were transferred to PVDF membranes for 2 h at 70 mA. Membranes were then blocked for 2 h with Tween 20‐Tris Buffered Saline (TTBS) containing albumin 4% and incubated with anti‐p‐ERK, anti‐total ERK, anti‐P‐CREB, or anti‐total CREB and anti‐β actin for 2 h. After washing several times with TTBS, the corresponding secondary antibodies were added for 45 min. Finally, the membranes were revealed using the Western Blotting Detection Kit (Thermo Fisher Science) and analyzed and quantified using Scion‐Image software (Scion Corporation Informer Technologies Inc, Meyer Instruments Inc., Houston, EEUU).
3
Apoptosis Protein Expression Analysis
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We evaluated the expression levels of proteins associated with apoptosis using Western blotting. Several antibodies were used, including rabbit anti-Bax antibody (1:1000, Abcam, Waltham, MA, USA), rabbit anti-Bcl2 antibody (1:1000, Abcam, Waltham, MA, USA), rabbit anti-PARP antibody (1:2000, Abcam, Waltham, MA, USA), and rabbit anti-caspase-3 antibody (1:2000, Abcam, Waltham, MA, USA). As a final step, the protein bands were observed using a Western blotting detection kit (Thermo, Waltham, MA, USA) after being treated with a HRP-conjugated goat anti-rabbit IgG antibody (1:1000, Abcam, Waltham, MA, USA). In order to determine protein expression levels, an image analyzer was used (ATTO, Tokyo, Japan).
4
Protein Extraction and Western Blotting in 3T3-L1 Cells
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For protein extraction, 3T3-L1 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors and then centrifuged. All processes were carried out in ice conditions. The protein concentration of extracts was measured using PierceTM BCA protein assay (Thermo Fisher Scientific; Boston, MA, USA). Proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Herculues, CA, USA). PVDF membranes were blocked with 5% BSA and incubated overnight with primary antibodies (1:1000) against PPARγ, C/EBPα, FABP4, C/EBPβ, cyclin D1, cyclin B1, cyclin-dependent kinase 6 (CDK6), CDK2, Akt, p-Akt, SOD1, catalase, phosphor-AKT, AKT, phosphor-ERK1/2, ERK1/2, phosphor-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, and β-actin (Cell Signaling Technology; Danvers, MA, USA). After washing in TBS with Tween 20, the membranes were incubated with secondary antibodies (1:2000 dilution). The band signals were detected using enhanced chemiluminescence (ECL; Thermo Fisher Scientific) Western blotting detection kit. The reference protein is β-actin, which has no difference in expression levels between preadipocytes and adipocytes (Figure S1 ).
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