Tissues were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After blocking with 3% bovine serum albumin (BSA; Servicebio, Wuhan, China), the slides were incubated with primary antibodies overnight at 4°C. Primary antibody concentrations were as follows: anti-PFN1, 1:100 (Abcam), anti-Napsin A, 1:100 (Abcam), anti-ROCK1, 1:100, (ImmunoWay, Plano, TX, United States), anti-ROCK2, 1:100 (ImmunoWay) and anti-annexin A1, 1:100 (Servicebio). Next, the slides were incubated with fluorophore-conjugated secondary antibodies (1:5,000, Proteintech, Wuhan, China) at room temperature. DAPI (Servicebio) was used to stain the nuclei. The samples were then visualized using a fluorescence microscope (Nikon, Tokyo, Japan).
Anti pfn1
Manufactured by Abcam
Sourced in United Kingdom
Anti-PFN1 is a laboratory reagent used to detect and quantify the Profilin 1 (PFN1) protein, which is involved in the regulation of actin cytoskeleton dynamics. This antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and ELISA, to measure the expression levels of PFN1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Nikon (1 mentions)
Fluorophore conjugated secondary antibody, by Proteintech (1 mentions)
Bovine serum albumin (bsa), by Wuhan Servicebio Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
β actin, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti t akt, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Anti p akt, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti fak, by Merck Group (1 mentions)
Anti perk1 2, by Merck Group (1 mentions)
Anti pfn1, by Merck Group (1 mentions)
Anti pfak, by Merck Group (1 mentions)
Anti smad3, by Merck Group (1 mentions)
Anti erk1 2, by Merck Group (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti p smad3, by Merck Group (1 mentions)
4 protocols using anti pfn1
1
Immunofluorescence Staining of Key Proteins
2
Whole Cell Lysate Protein Extraction and Western Blot Analysis
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Whole lysate protein was extracted from cells using a radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. In this process, 15 μg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a 0.2-μm-pore PVDF membrane (Millipore). The membrane was blocked with 5% bovine serum albumin/Tris-Buffered Saline, 0.1% Tween 20 for 1 hour and then incubated with specific primary antibody at 4°C overnight against anti-PFN1 (1:2000; Abcam, Cambridge, UK) and β-actin (1:2000, Abcam), followed by incubation with mouse or rabbit immunoglobulin G-horseradish peroxidase (1:3000, Abcam). Protein bands were visualized using a Lumi Femto solution (Dogen, Seoul, Korea).
3
Western Blot Analysis of PFN1, AKT, and GAPDH
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Cell and tissue lysates were prepared by RIPA Lysis Buffer (Beyotime, Shanghai, China). Proteins were separated and transferred to PVDF membrane (Bio-Rad, USA). After incubations with anti-PFN1 (1:1000, abcam, UK), anti-p-AKT (1:1000, abcam, UK), anti-t-AKT (1:1000, abcam, UK) and anti-GAPDH (1:1000, abcam, UK) overnight, the membrane had incubation with goat anti-rabbit IgG secondary antibody and detection through chemiluminescence. GAPDH was a reference.
4
Protein Expression Analysis in 2D and 3D Cultures
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Total cell lysate from 2D monolayer culture was prepared by extracting cells with modified RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) supplemented with 50 mM NaF, 1 mM sodium pervanadate, and protease inhibitors. Total cell lysate from 3D culture was prepared by incubating cells at 4 °C in the modified RIPA buffer supplemented with 0.5% SDS and protease/phosphatase inhibitor cocktail (Pierce). The extracts were clarified by centrifugation at 13,000 rpm for 30 min and the supernatant was used for immunoblotting. Sources of different antibodies were: anti-Pfn1 (Abcam), anti-phospho-FAK (Y397) (Invitrogen), anti-Smad3 (Biorad), anti-ERK1/2, anti-phospho-ERK1/2 and anti-phospho-Smad3 (S423/S425) (Cell Signalling), anti-Pfn2 and anti-FAK (Santa Cruz), and anti-GAPDH and anti-tubulin (Sigma-Aldrich) Immunoblotting concentrations for different antibodies were: 1:3000 for anti-Pfn1, anti-GAPDH and anti-tubulin; 1:500 for anti-Pfn2, anti-ERK1/2, anti-pERK1/2 and anti-FAK; and 1:1000 for anti-Smad3, anti-pSmad3 and anti-pFAK.
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