Immunoblotting was conducted according to the earlier study [27 (link)]. Briefly, WEHI-3 cells were grown in 75 cm2 culture flask and then treated with ZnO-NPs. After incubation for 24, 48, and 72 hours, respectively, harvested cells were washed twice with ice-cold PBS. Then, total cell proteins were isolated using a RIPA lysis buffer, separated on an SDS-PAGE, and transferred to PVDF membranes. After being blocked, the membranes were incubated with anti-Bcl-2 (Santa Cruz, CA, USA) and anti-Bax (Santa Cruz, CA, USA) (1 : 1000) at 4°C overnight. After washing for 30 minutes at 10 minute intervals, the membranes were loaded with an HRP secondary antibody (Goat polyclonal to rabbit IgG, AB97051, Abcam, USA) (1 : 2000) at room temperature for 1 hour. Finally, specific protein bands were detected using chemiluminescence (ECL) detection kit (Abcam, USA). β-actin was used as the internal control (Santa Cruz, CA, USA).
Ecl detection kit
Manufactured by Abcam
Sourced in United States
The ECL detection kit is a reagent system used for the chemiluminescent detection of proteins in Western blot analysis. It provides a sensitive and quantitative method for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ab97051, by Abcam (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti bax, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ipp 6, by Media Cybernetics (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Pvdf membrane, by Santa Cruz Biotechnology (1 mentions)
Pierce rapid gold bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Protein extraction kit, by Beyotime (1 mentions)
4 protocols using ecl detection kit
1
Immunoblotting Protocol for Bcl-2 and Bax
2
Immunoblotting Analysis of Osteoblast Proteins
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Proteins from whole cell lysates were extracted using a radio-immunoprecipitation assay. Osteoblasts stimulated with rhHMGB1 were lysed in lysis buffer. Supernatants were prepared by centrifugation, electrophoresed on a 10% SDS/PAGE and blotted on to a polyvinylidene difluoride membrane. Immunoblotting was performed using antibodies specific to TLR2, TLR4 and GAPDH, followed by an horseradish peroxidase (HRP)-conjugated secondary antibody and developed using an ECL detection kit (Abcam). The relative amounts of protein bands on the blots were determined using IPP 6.0 software (Media Cybernetics Inc).
3
Validation of Protein Extraction from FFPE Tissues
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Western blot was performed to validate the success of the protein extraction from the FFPE tissues. Protein extracts were separated on SDS denaturing 10% polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride membranes (PVDF). The membranes were blocked with 5% skim milk for 1 h, washed, and then probed with primary antibodies raised against actin and Akt-1 (Abcam) proteins at 4 °C overnight. Finally the membranes were treated with HRP-coupled secondary antibodies for 1 h, and the membranes were then washed twice with TBST (1×) for 30 min each thereafter. Protein detection is revealed using the ECL detection kit (Abcam plc, 330 Cambridge Science Park, Cambridge, UK). Blot images were obtained with the Image Lab Software (BioRad, Chemidoc imaging instrument).
4
Quantitative Western Blot Analysis of EMT Markers
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Total protein from ARPE19 cells or retinas of diabetic mice was harvested by Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China) and quantitated by Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Samples were analyzed on the sodium dodecylsulfate (SDS)-polyacrylamide gels electrophoresis (PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Santa Cruz Biotech, Dallas, TX, USA). 5% bovine serum albumin (BSA; Thermo Scientific, Waltham, MA, USA) was used for blocking, and primary rabbit polyclonal antibodies purchased from Abcam (Abcam, Cambridge, MA, USA) including SOX4 (1:800), E-cadherin (1:800), N-cadherin (1:1200), Snail (1:1000) and Vimentin (1:1000) were applied onto the PVDF membranes at 4 °C for overnight incubation, respectively. The internal reference was a primary rabbit polyclonal antibody against GADPH (1:2500; Abcam, Cambridge, MA, USA). After washing, a horseradish-peroxidase-conjugated against rabbit antibody (1:2000; Abcam, Cambridge, MA, USA) was applied onto the PVDF membranes at room temperature for 1 h. An ECL detection kit (Abcam, Cambridge, MA, USA) was used to detect the results. Image J software (NIH, Bethesda, Maryland, USA) was used to calculated the grey level ratio of target protein to internal reference.
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