The total protein in transfected KYSE450 and KYSE510 cells was extracted and quantified with the RIPA buffer (Solarbio, China), which was supplemented with 1% proteinase inhibitor cocktail. The protein concentration was obtained with the Pierce BCA protein assay kit (Thermo Fisher Scientific, USA). Next, the quantified protein was loaded into 10% or 12% SDS-PAGE for electrophoresis separation. The separated protein bands were then electronically transferred to the membranes (Sigma-Aldrich, USA). Following that, the membranes were put under ice for 2 h with 5% BSA at room temperature. This step was followed by the incubation of the membranes overnight with primary antibodies at 4 °C and 1.5 h incubation with secondary detection antibody (Cat# ab205718, Abcam, USA) at room temperature. Finally, the protein immunoblots were visualized using the Enhanced Chemiluminescent (ECL) Reagent Kit (Thermo Fisher Scientific. USA). The density of the blots was obtained using ImageJ software. All the primary antibodies were purchased from Abcam (USA), including anti-CyclinB1 (Cat# ab32053), anti-ICAM1 (Cat# ab53013), anti-VCAM1 (Cat# ab134047), anti-Cleaved PARP (Cat# ab32561), anti-Bax (Cat# ab32503), anti-Cleaved Caspase-3 (Cat# ab2302), anti-TXNRD1 (Cat# ab124954) and anti-β-actin (Cat# ab8227).
Secondary detection antibody
Manufactured by Abcam
Sourced in United States
Secondary detection antibodies are used to amplify and detect the primary antibody signal in immunoassays and other immunodetection techniques. They bind to the constant region of the primary antibody, allowing for the visualization and quantification of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Anti icam 1, by Abcam (2 mentions)
Anti cyclin b1, by Abcam (2 mentions)
Anti vcam 1, by Abcam (2 mentions)
Anti txnrd1, by Abcam (2 mentions)
Anti cleaved parp, by Abcam (2 mentions)
Anti cleaved caspase 3, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Ripa buffer, by Solarbio (2 mentions)
Anti bax, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescent ecl reagent kit, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
3 protocols using secondary detection antibody
1
Protein Expression Analysis in Esophageal Cancer Cells
2
PEDV Infection Detection in IPEC-J2 Cells
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IPEC-J2 cells were seeded in a 12-well plate at a density of 5 × 105 cells/mL until 70% confluence. After 24 h of PEDV infection, the cells were washed thrice with PBS and fixed in 4% formaldehyde (Nanjing Novezan Biotechnology Co., Ltd., Nanjing, China) at room temperature for 60 min. Then, 0.5% Triton X-100 (Nanjing Novezan Biotechnology Co., Ltd., Nanjing, China) was added, and the mixture was incubated for 15 min, 5% BSA (Nanjing Novezan Biotechnology Co., Ltd., Nanjing, China) was subsequently added and the reaction was allowed to proceed for 2 h. The supernatant was discarded, followed by the addition of a primary antibody (Veterinary Medical Research & Development Co., Ltd., Washington, DC, USA) (1:500) and secondary detection antibody (Abcam Co., Ltd., Cambridge, UK) (anti-mouse IgG, 1:200). The cells were then stained with DAPI (Abcam Co., Ltd., Cambridge, UK) (1:800) and observed under a fluorescence microscope.
3
Protein Analysis of Transfected Esophageal Cancer Cells
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The total protein in transfected KYSE450 and KYSE510 cells was extracted and quanti ed with the RIPA buffer (Solarbio, China), which was supplemented with 1% proteinase inhibitor cocktail. The protein concentration was obtained with the Pierce BCA protein assay kit (Thermo Fisher Scienti c, USA). Next, the quanti ed protein was loaded into 10% or 12% SDS-PAGE for electrophoresis separation. The separated protein bands were then electronically transferred to the membranes (Sigma-Aldrich, USA). Following that, the membranes were put under ice for 2 h with 5% BSA at room temperature. This step was followed by the incubation of the membranes overnight with primary antibodies at 4°Cand 1.5-hour incubation with secondary detection antibody (Cat# ab205718, Abcam, USA) at room temperature. Finally, the protein immunoblots were visualized using the Enhanced Chemiluminescent (ECL) Reagent Kit (Thermo Fisher Scienti c. USA). The density of the blots was obtained using ImageJ software. All the primary antibodies were purchased from Abcam (USA), including anti-CyclinB1 (Cat# ab32053), anti-ICAM1 (Cat# ab53013), anti-VCAM1 (Cat# ab134047), anti-Cleaved PARP (Cat# ab32561), anti-Bax (Cat# ab32503), anti-Cleaved Caspase-3 (Cat# ab2302), anti-TXNRD1 (Cat# ab124954) and anti-β-actin (Cat# ab8227).
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