Protein samples of 5 μg were dissolved in sample buffer (Laemmli buffer, Bio-Rad) containing DTT (dithiothreitol) and treated for 5 min at 95 °C. Protein samples were separated on a 4–12% CRIT XT BIS-TRIS GEL (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad). Membranes were blocked with 5% fat-free milk in tris-buffered saline containing 0.1% Tween-20 for 1.5 h. Primary antibodies were added and membranes were incubated overnight at 4 °C. Hereafter, an adequate horseradish peroxidase (HRP)-conjugated secondary antibodies were added and incubated for 2 h at room temperature. Visualization was performed using Clarity ECL or Clarity ECL max (Biorad). Primary Antibodies: Klotho (MAB1819, 1:500, R&D), KIM-1 (#3809, 1:1000, ProSci, Poway, CA, USA), Pan-Actin antibody (#4968, dilution, Cell Signaling, Frankfurt am Main, Deutschland).
Pan actin antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Pan-actin antibody is a laboratory tool used to detect and quantify actin proteins in various samples. Actin is a highly abundant and ubiquitous cytoskeletal protein found in all eukaryotic cells. The Pan-actin antibody recognizes multiple isoforms of actin, making it a versatile tool for research applications involving the analysis of actin expression and distribution.
Automatically generated - may contain errors
Lab products found in correlation
Klotho, by R&D Systems (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Clarity ecl, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
G actin f actin in vivo assay kit, by Cytoskeleton (1 mentions)
Peroxidase conjugated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Sds sample buffer, by Boston BioProducts (1 mentions)
0.45 m nitrocellulose membrane, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
5 protocols using pan actin antibody
1
Western Blot Analysis of Kidney Injury Biomarkers
2
Immunoblotting for MuRF1 and Actin
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Antibody against MuRF1 was purchased from ECM biosciences (MP3401) and pan-actin antibody from cell signaling (#12748).
3
Quantifying Cardiac Myofibroblast F-Actin Levels
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Cardiac myofibroblasts were cultured and treated in 10 cm dishes, and harvested at ~ 50–60% confluency. Cells were washed twice in pre-warmed 1X PBS to remove any excess medium and serum. After removing as much PBS as possible, F and G actin were isolated using a G-actin/F-actin in vivo Assay Kit (Cytoskeleton Inc., Denver, CO; #BK037), as per the manufacturer’s directions. Immunoblotting was performed with pan-actin antibody (1:1000; Cell Signaling; #4968), and peroxidase-conjugated goat anti-rabbit secondary antibodies (1:10 000; Jackson ImmunoResearch).
4
Notch3 and TRPC6 Protein Expression Analysis
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Lung tissues harvested from mice were homogenized with radioimmunoprecipitation
(RIPA) buffer, followed by protein isolation. The samples were diluted with
6 × SDS-sample buffer (Boston BioProducts, USA), heated for 10 min at 95°C, and
loaded on 10% SDS polyacrylamide gels. The protein samples were separated by
electrophoresis and transferred to a 0.45 µm nitrocellulose membrane (BioRad,
USA). The membrane was blocked with 5% bovine serum albumin (Sigma) in
Tris-Buffered Saline with Tween 20 (TTBS) for an hour at room temperature and
then incubated overnight at 4°C with anti-Notch3 (VMA00484, 1:1000, Bio-Rad) or
anti-TRPC6 (bs-2393R, 1:1000, Bioss) primary monoclonal antibody. The membrane
was washed with 1X Tris-Buffered Saline, 0.1% Tween® 20 Detergent (TBST) and
then incubated for an hour at room temperature with the secondary anti-mouse or
anti-rabbit IgG, Horseradish peroxidase (HRP)-linked antibody (1:5000; Cell
Signaling). The membrane was subsequently developed after adding substrate
(Thermo Fisher Scientific). All membranes were probed for Pan-Actin antibody
(Cat# 4968S, 1:2000, Cell Signaling) or β-actin antibody (Cat# sc-47778, 1:1500,
Santa Cruz Biotechnology) as internal controls. Band intensities on the membrane
were quantified using Image J software.
(RIPA) buffer, followed by protein isolation. The samples were diluted with
6 × SDS-sample buffer (Boston BioProducts, USA), heated for 10 min at 95°C, and
loaded on 10% SDS polyacrylamide gels. The protein samples were separated by
electrophoresis and transferred to a 0.45 µm nitrocellulose membrane (BioRad,
USA). The membrane was blocked with 5% bovine serum albumin (Sigma) in
Tris-Buffered Saline with Tween 20 (TTBS) for an hour at room temperature and
then incubated overnight at 4°C with anti-Notch3 (VMA00484, 1:1000, Bio-Rad) or
anti-TRPC6 (bs-2393R, 1:1000, Bioss) primary monoclonal antibody. The membrane
was washed with 1X Tris-Buffered Saline, 0.1% Tween® 20 Detergent (TBST) and
then incubated for an hour at room temperature with the secondary anti-mouse or
anti-rabbit IgG, Horseradish peroxidase (HRP)-linked antibody (1:5000; Cell
Signaling). The membrane was subsequently developed after adding substrate
(Thermo Fisher Scientific). All membranes were probed for Pan-Actin antibody
(Cat# 4968S, 1:2000, Cell Signaling) or β-actin antibody (Cat# sc-47778, 1:1500,
Santa Cruz Biotechnology) as internal controls. Band intensities on the membrane
were quantified using Image J software.
5
Western Blot Analysis of Cardiac Proteins
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Proteins from RV tissue were isolated, and Western blot analysis was performed according to protocols described previously [9] . The following antibodies were used for Western blotting: CILP1 antibody (Biorbyt, Cambridge, UK; catalogue no. orb182643), atrial natriuretic peptide (ANP) antibody (Merck, Kenilworth, NJ, USA; catalogue no. AB5490), and pan-actin antibody (Cell Signaling Technology, Danvers, MA, USA; catalogue no. 4968).
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