Indirect competitive ELISA was used to test the specificity and presence of antibodies, as described before. ENR-BSA dissolved in 0.5 M carbonate buffer (100 ng per well, 100 μL) was used to coat the microplates at 4°C for 12 h, and 1% casein in PBS (w/v, 250 μL/well) was used to wash and block the plates, and incubated for 60 min at 37°C. The blocking solution was carefully removed, and the competitor in PBS buffer or PBS buffer was added to each well, and incubated for 60 min at room temperature. PBS was used to wash the plates 3 times, and 100 μL of anti-mouse IgG-HRP conjugate at a dilution of 1 1000 in 1% skim milk (Boster, China) was used to incubate the plates for 60 min. We used 100 μL of TMB solution and washing buffer to wash the plates for 5 min. To halt the color development, we used 100 μL of 2N H2SO4, and a microtiter plate reader (Thermo Scientific, MA) was used to detect the plates based on the absorbance at 450 nm, according to the manufacturer’s protocol.
Skim milk
Manufactured by Boster Bio
Sourced in China
Skim milk is a laboratory product that is used as a blocking agent in various immunoassay techniques, such as Western blotting and ELISA. It is made by removing the fat content from whole milk, leaving behind only the protein and carbohydrate components. Skim milk can be used to reduce non-specific binding of antibodies or other proteins to the solid support in these assays, thereby improving the signal-to-noise ratio and specificity of the results.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Microtiter plate reader, by Thermo Fisher Scientific (1 mentions)
Bio spectrum gel imaging system, by Analytik Jena (1 mentions)
Pvdf membrane, by Boster Bio (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Ab50917, by Abcam (1 mentions)
Ecl detection kit, by Merck Group (1 mentions)
Ab8227, by Abcam (1 mentions)
4 protocols using skim milk
1
Antibody Specificity Evaluation by ELISA
2
SDS-PAGE Protein Extraction and Detection
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Total protein samples were extracted from kidney tissues and NRK-52E cells using appropriate cold lysis buffer containing 1 mM PMSF following the manufacturer’s instructions. After determination of the contents, the proteins were separated by SDS-PAGE (8%–12%), transferred to PVDF membranes (Millipore, MA, USA), then blocked with 5% dried skim milk (Boster Biological Technology, Wuhan, China) and incubated overnight at 4 °C with the primary antibodies listed in Table 2 . The membranes were incubated with an HRP-conjugated secondary antibody for 2 h at room temperature. And protein detection was performed based on an enhanced chemilumines-cence (ECL) method and photographed by using a Bio-Spectrum Gel Imaging System (UVP, Upland, CA, USA). Intensity values of the relative protein levels were normalized to GAPDH.
3
Dioscin-induced Protein Expression Analysis in HeLa and SiHa Cells
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HeLa and SiHa cells were treated with dioscin (1.25, 2.5 and 5.0 μg/mL), and protein samples were extracted using cell lysis buffer (Beyotime, Shanghai, China). An aliquot (50 μg protein) was loaded onto a 8%–12% SDS-PAGE gel and separated electrophoretically. Then the target proteins were transferred to a PVDF membrane (Millipore, MA, USA). After blocking in 5% dried skim milk (Boster Biological Technology, Wuhan, China), the PVDF membrane was incubated with primary antibodies (listed in Table S1 ), respectively. Then the membrane was incubated with horseradish peroxidase-conjugated antibodies at a 1:5000 dilution. Protein detection was performed based on an enhanced chemiluminescence (ECL) method and photographed by using a BioSpectrum Gel Imaging System (HR410, UVP, Upland, CA, USA). In order to eliminate the variations, data were adjusted to GAPDH expression: IOD of objective protein versus IOD of GAPDH expression.
4
Western Blot Analysis of Cell Lysates
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Cells were transfected for 48 h and lysed using RIPA buffer (catalog# 9806S, CST, Danvers, MA, USA). The samples were centrifuged, and proteins in the supernatants were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with 10% or 12% gels and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked by incubation with skim milk(1:5000; Boster, Wuhan, China) and then incubated for 12 h at 4°C with primary antibodies against E2F3 (catalog# ab50917; Abcam, Cambridge, UK), PRDM1 (catalog# ab106766, Abcam), cyclin-dependent kinase 17 (CDK17, catalog# ab159068, Abcam), or β-actin (catalog# ab8227, Abcam). After washing, the membranes were incubated for 2 h at 37°C with anti-rabbit, anti-mouse, or anti-goat IgG secondary antibodies. Electronic chemical Laboratory (ECL) detection kit was used for the signal development (Merck Millipore, Darmstadt, Germany).
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