The proliferation of G1E-ER4 cells was assessed by using a BrdU cell proliferation assay kit, according to the manufacturer’s instructions (Cell Signaling Technology, #6813). We plated 1 × 104 G1E-ER4 cells into wells of a 96-well plate, added 10 μl 1x BrdU into each well and incubated for 4, 8 or 24 h. After the incubation, the cells were fixed and denatured for 30 min, then 100 μl/well BrdU detection antibody solution (Cell Signaling Technology, #94079, 1:100 dilution) was added and incubate at room temperature for 1 h. After the incubation, each well was washed three times with 100 μl wash buffer. Next 100 μl/well HRP-conjugated secondary antibody solution (Cell Signaling Technology, #34709, 1:100 dilution) was added and incubated at room temperature for 30 min. After the incubation, the cells were washed three times with 100 μl wash buffer. TMB substrate (100 μl) was added and incubated for 30 min before adding stopping buffer. After adding the stopping buffer, absorbance was read at 450 nm to detect cell proliferation.
Hrp conjugated secondary antibody solution
Manufactured by Cell Signaling Technology
Sourced in United States
HRP-conjugated secondary antibody solution is a reagent used in immunodetection techniques. It contains a horseradish peroxidase (HRP) enzyme conjugated to a secondary antibody that binds to the primary antibody. The HRP enzyme can catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and visualization of the target protein.
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Lab products found in correlation
2 protocols using hrp conjugated secondary antibody solution
1
Quantifying G1E-ER4 Cell Proliferation
2
Apoptosis Pathway Protein Expression
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After treatment with BSGLEE for 48 h, cells were washed twice with PBS and fully lysed with ice-cold RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA). Then cell lysates were centrifuged at 13,000 rpm for 10 min at 4°C. Supernatants were collected and concentration of total protein was quantified by BCA assay. Protein samples (40 µg/lane) were separated by 10–12% SDS-PAGE gel and then transferred to polyvinylidene fluoride membranes (PVDF) (Merck Millipore, Darmstadt, Germany) using a transfer system (Bio-Rad Laboratories, 100 v, 120 min). After blocking with 5% skim milk in TBST for 1 h at RT, the membranes were incubated overnight at 4°C in the primary antibody solution against pro-caspase-3, cleaved caspase-3, pro-caspase-7 and PARP antibodies according to the manufacturers instruction. Membranes were washed 3 times with TBST for 10 min and incubated in the HRP-conjugated secondary antibody solution (Cell Signaling Technology, Inc., Beverly, MA, USA) for 1 h at room temperature. The signals were detected using ECL chemiluminescence reagent (Perkin-Elmer) and β-actin was used as loading control.
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