Western blots on cell lysates were performed as previously described [24 (link)]. Visualization and quantification were performed with Odyssey Infrared Imaging System (Li-Cor Biosciences). Experiments were repeated at least three times. PTEN antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA). Actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary goat anti-rabbit IgG IRDye antibody was purchased from LI-COR Biosciences (Lincoln, NE). Secondary mouse IgM IRDye antibody was purchased from Rockland Immunochemicals Inc. (Gilbertsville, PA).
Actin antibodies
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Actin antibodies are laboratory reagents used to detect and quantify the presence of actin, a ubiquitous and essential cytoskeletal protein found in all eukaryotic cells. These antibodies are designed to specifically bind to actin, allowing researchers to study its distribution, localization, and regulation within cells.
Automatically generated - may contain errors
Lab products found in correlation
Secondary goat anti rabbit igg irdye antibody, by LI COR (2 mentions)
Pten antibody, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Secondary mouse igm irdye antibody, by Rockland Immunochemicals (2 mentions)
Ampk and acc antibody sampler kit, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Ser473 akt, by Cell Signaling Technology (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Anti traf6, by Abcam (1 mentions)
Igg1 pe, by R&D Systems (1 mentions)
Hrp conjugated rabbit and mouse secondary antibodies, by GE Healthcare (1 mentions)
Taci pe, by R&D Systems (1 mentions)
Igg1 fitc, by BD (1 mentions)
Ha hrp, by Roche (1 mentions)
P mtor s2481, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Rotenone, by Merck Group (1 mentions)
Succinate, by Merck Group (1 mentions)
2 6 dichloroindophenol, by Merck Group (1 mentions)
Sulforhodamine b srb, by Merck Group (1 mentions)
12 protocols using actin antibodies
1
PTEN Quantification in Cell Lysates
2
Quantitative Western Blot Analysis
3
Western Blot Antibody Reagents
4
Mitochondrial Respiratory Chain Profiling
5
Investigating IGFBP-3 and LRP1 Signaling Pathways
6
Immunoblotting of Phosphorylated ZAP70
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TIL14 were mixed at a 5:1 cell ratio with ice cold 624mel ADAR1-KD or Scramble cells. Following gentle vortex stimulations were carried for 10 min using a water-bath at 37°C and terminated with the addition of cold PBS. Cells were immediately pelleted and lysed by triton based lysis buffer supplemented with phosphatase and protease inhibitor cocktails for 30 min on ice. 20 μg of protein from the lysates were used for subsequent Western Blot (WB), as previously described [21 (link)]. Membranes were exposed to p-ZAP70Y319 primary antibody (Cell Signaling Technology, cat#C-2701S) overnight at 4°C. For total ZAP70 and load control analysis, membranes were striped by low pH buffer, blocked and exposed to ZAP70 (Santa Cruz Biotechnology, cat#sc-574) and actin antibodies (Santa Cruz Biotechnology, cat#sc-1616) for 60 min at room temperature. Exposure was done by a secondary peroxidase-conjugated anti-rabbit Ab (Santa Cruz Biotechnology, cat#SC-1616R) and standard ECL reagent (Pierce, cat#PIR-34077). Revelation and quantification of WB data was performed using an ImageQuant LAS 500 imager (GE Healthcare) and the image analysis program Image Studio Digits (LI-COR, Lincoln, NE).
7
Western Blot Analysis of Protein Markers
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For extraction of protein, cells were rinsed with ice-cold phosphate buffered saline (PBS) and then lysed in ice-cold RIPA buffer (containing protease inhibitor cocktail and phosphatase inhibitor). Extracted protein were separated through electrophoresis, the proteins were transferred to nitrocellulose membranes by electrophoretic transfer. The membranes were blocked in 6% bovine serum albumin for 1 h, rinsed and then incubated with COX-2, iNOS, NF-κB, PARP, GAPDH, pIκBα, or actin antibodies (Santa Cruz, CA) for 1 h at room temperature. After wash the membrane, the membranes were incubated for 30 min with horse radish peroxidase-conjugated secondary antibodies. The protein levels were visualized by an enhanced chemiluminescence assay according to the manufacturer’s instructions (Amersham Corp. Newark, NJ).
8
Western Blot Analysis of Protein Expression
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Confluent whole cells were lysed to generate total protein for separation by 8-10% SDS polyacrylamide gels, transferred onto nitrocellulose membranes, and analyzed with antibodies against: NHE1 (BD Transduction Laboratories), vimentin (Santa Cruz Biotechnologies), or β-catenin (Cell Signaling Technology). β-tubulin (Sigma) or actin antibodies (Santa Cruz Biotechnologies) were used as a loading control. Densitometry using the program Image J (ImageJ 1.48v software, National Institutes of Health, Bethesda, MD) was used to quantify expression of the protein of interest versus the loading control.
9
Protein Extraction and Western Blotting
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Cells were lysed
in RIPA buffer (with
1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease
inhibitor mixture) and centrifuged at 4 °C for 10 min
at 14,000g. The supernatant was boiled in Laemmli
sample buffer for 10 min and subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis and transferred to Immobilon-P
filters (Millipore). The filters were blocked for 1 h with PBS containing
5% nonfat dry milk and 0.1% Tween 20 and incubated with primary and
secondary antibodies, and the filters were developed using SuperSignal
reagent (Thermo Scientific). MDM2 was detected using monoclonal antibody
3G9. Actin antibodies were purchased from Santa Cruz Biotechnology.
DO-1 for human p53 and p21 antibody were from BD Pharmingen.
in RIPA buffer (with
1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease
inhibitor mixture) and centrifuged at 4 °C for 10 min
at 14,000g. The supernatant was boiled in Laemmli
sample buffer for 10 min and subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis and transferred to Immobilon-P
filters (Millipore). The filters were blocked for 1 h with PBS containing
5% nonfat dry milk and 0.1% Tween 20 and incubated with primary and
secondary antibodies, and the filters were developed using SuperSignal
reagent (Thermo Scientific). MDM2 was detected using monoclonal antibody
3G9. Actin antibodies were purchased from Santa Cruz Biotechnology.
DO-1 for human p53 and p21 antibody were from BD Pharmingen.
10
PTEN Quantification in Cell Lysates
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