The cell lysates were both centrifuged at 13,000 rpm (at 4°C for 5 min). After that 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) were used to segregate them and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The filters were blocked with blocking buffer (Sangon Biotech) for 45 min. Membranes were incubated with primary antibodies anti-LRH1 (Abcam, ab223211), anti-E-cadherin (Abcam, ab1416), anti-N-cadherin (Abcam, ab18203), anti-Vimentin (Abcam, ab92547), anti-β-catenin (Cell Signaling Technology, D10A8)) at 4°C overnight and with secondary antibodies at 1:5,000 dilution at room temperature for 2 h, followed by incubating with β-actin (Abcam, ab179467) at room temperature for 1.5 h.
Blocking buffer
Manufactured by Sangon
Sourced in United States
Blocking buffer is a laboratory solution used to prevent non-specific binding in various immunoassays, such as Western blotting and enzyme-linked immunosorbent assays (ELISAs). It contains a mixture of proteins, surfactants, and other additives that help to block unoccupied binding sites on the solid support, reducing the likelihood of non-specific interactions between the target analyte and the solid support.
Automatically generated - may contain errors
Lab products found in correlation
Ab223211, by Abcam (2 mentions)
Ab179467, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab92547, by Cell Signaling Technology (1 mentions)
Ab18203, by Abcam (1 mentions)
Ab1416, by Abcam (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg, by Merck Group (1 mentions)
Rabbit anti cldnd1, by Abcam (1 mentions)
Hoechst 33258, by Sangon (1 mentions)
Normal rabbit igg, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Sangon (1 mentions)
3 protocols using blocking buffer
1
Western Blot Analysis of EMT Markers
2
Immunofluorescence Staining of CLDND1
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When the coverage of cells on cover slips reached about 90%, cells were fixed for 15 minutes at room temperature in 4% paraformaldehyde. After aspiration of fixative, samples were rinsed three times in 1×PBS for 5 minutes each and blocked in Blocking Buffer (1×PBS supplemented with 0.3% Triton X-100 (Sangon Biotech) and 5% normal goat serum (Life Technologies)) for 60 minutes. After incubation with the primary antibody (1:100) overnight at 4℃, samples were rinsed three times in 1×PBS for 5 minutes each and incubated with the anti-rabbit IgG second antibody (1:500) for 60 minutes at room temperature in dark. The normal rabbit IgG (Life Technologies) was used as negative control. The antibodies for immunofluorescence were as follows: rabbit anti-CLDND1 (Abcam), rabbit anti-IgG (Merck Millipore), and anti-rabbit IgG (Alexa Fluor® 488 Conjugate) (Life Technologies). The nuclei was labeled using Hoechst 33258 (Sangon Biotech), and cells were visualized using a fluorescence microscope Ti-S (Nikon, Tokyo, Japan).
3
Western Blot Analysis of LRH1 Protein
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The cell lysates were both centrifuged at 13000 rpm (at 4℃ for 5 minutes). After that 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to segregate them, and then transferred onto polyvinylidene di uoride (PVDF) membranes (Millipore, Bedford, MA, USA).The lters were blocked with blocking buffer (Sangon Biotech) for 45 min.Membranes were incubated with Primary antibodies against LRH1 (Abcam, ab223211) at 4℃ overnight, and with secondary antibodies at 1:5000 dilution at room temperature for 2 h, following by incubating with β-actin (Abcam, ab179467) atroom temperature for 1.5 h.
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