Following cell lysis, protein concentration was assessed using the Bio-Rad DC Protein Assay (Bio-Rad:500–0111) and protein (2–40 μg) was resolved by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PDVF) membranes (Immunobilon Transfer Membranes:IPVH00010). Transferred membranes were blocked with 5% milk for 30 minutes and probed with appropriate antibody in 1% milk solution for either 1 hour at room temperature or 12 hours at 4°C, with three 10 min washes with 1XPBS-0.05% Tween 20 between each antibody incubation. Antibodies included: Anti-Stau1 [1:1000] (Abcam:ab73478), Anti-GAPDH [1:10,000] (Abcam:ab8245), Anti-β-Actin [1:500] (Santa Cruz:sc-47778), Anti-CUGBP1 [1:1000] (Santa Cruz:sc-20003), Anti-hnRNP H [1:5000] (Abcam:10374), Anti-MyoD [1:300] (BD Pharmingen: 554130), Anti-MBNL1 antibody [1:300] (Abnova:H00004154), Anti-HA F7 probe [1:1000] (Santa Cruz:sc-7392). Secondary antibodies included: Mouse-anti-Rabbit HRP [1:20,000] (Jackson ImmunoResearch:211-032-171) and Goat-anti-Rabbit HRP [1:10,000] (Molecular Probes:MP 02764). Proteins on membranes were detected with Millipore-Luminata Crescendo Western HRP Substrate (WBLUR0500) and visualized on film (HyBlot CL Autoradiography Film:E3018).
Anti stau1
Manufactured by Abcam
Sourced in United States
Anti-STAU1 is a laboratory antibody product that recognizes the STAU1 (Staufen homolog 1) protein. STAU1 is an RNA-binding protein involved in various cellular processes. This antibody can be used for the detection and analysis of STAU1 in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cugbp1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti myod, by BD (1 mentions)
Goat anti rabbit hrp, by Thermo Fisher Scientific (1 mentions)
Mouse anti rabbit hrp, by Jackson ImmunoResearch (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti hsp90, by Proteintech (1 mentions)
Anti klf16, by Abcam (1 mentions)
Anti upf1, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by ZSGB-BIO (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Anti ppkr, by Abcam (1 mentions)
Anti pkr, by Cell Signaling Technology (1 mentions)
Anti p eif2α, by Cell Signaling Technology (1 mentions)
5 protocols using anti stau1
1
Western Blot Protein Detection
2
Western Blot Analysis of Protein Targets
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The cells were lysed in RIPA lysis buffer to collect the protein, which was quantified using the Micro BCA Protein Assay Reagent. The protein lysates (20 μg) were separated by 12% SDS poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose filter membrane NC, (Pall, Shanghai, China) with a pore size of 0. 22 μm. Membranes were blocked in PBST with 5% skimmed milk for 2 h in 37 °C. Following this, the primary antibodies (Anti-STAU1 (Abcam, Cambridge, MA, USA), Anti-RABV-N (1N1) (produced by lab)and Anti-GAPDH (Goodhere, Hangzhou, China)) were incubated at 37 °C for 2 h or 4 °C for overnight, washed thrice with PBST for 3–5 min, then incubate with secondary antibody coupled to horseradish peroxidase for 1 h at 37 °C. Membranes were washed again and analyzed by chemiluminescence using the ECL+ kit.
3
RNA-binding protein STAU1 and PPARγ Chromatin IP
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3T3-L1 cells (approximately 1 × 10 6 ) were added to RIP lysis buffer and RIP immunoprecipitation buffer, and incubated for 1 h at 4 °C. Then, 50 µl Beads (17-701; Millipore) and 10 µl anti-STAU1 (Abcam) were added and incubated overnight. The precipitates were washed 6 times with RIP washing buffer. Next, 150 µl proteinase K solution (17-701; Millipore) was added and incubated at 55 °C for 30 min, followed by washing with RIP washing buffer. Finally, after adding 400 µl of phenol: chloroform: isoprene mixture, RNA was extracted for PCR analysis.
Chromatin IP (ChIP)
Cells seeded on a 10-cm dish were cross-linked with 275 µl of 37% formaldehyde at room temperature for 10 min, and 1 ml glycine was then added and incubated at room temperature for 5 min to terminate the crosslinking. Cells were then lysed and sonicated to shear the chromatin to a manageable size. Next, 450 µl of ChIP dilution buffer, 2.25 µl protease inhibitor mixture, 20 µl of fully suspended beads, and 10 µl PPARγ antibody were added to each IP, and incubated overnight at 4 °C. DNA was extracted from the upper column of a 500-µl Bind Reagent A, which was added after cleaning the magnetic beads, and PCR analysis was then performed.
Chromatin IP (ChIP)
Cells seeded on a 10-cm dish were cross-linked with 275 µl of 37% formaldehyde at room temperature for 10 min, and 1 ml glycine was then added and incubated at room temperature for 5 min to terminate the crosslinking. Cells were then lysed and sonicated to shear the chromatin to a manageable size. Next, 450 µl of ChIP dilution buffer, 2.25 µl protease inhibitor mixture, 20 µl of fully suspended beads, and 10 µl PPARγ antibody were added to each IP, and incubated overnight at 4 °C. DNA was extracted from the upper column of a 500-µl Bind Reagent A, which was added after cleaning the magnetic beads, and PCR analysis was then performed.
4
Western Blot Analysis of Cellular Proteins
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Whole-cell protein extracts were prepared from cells (approximately 1 × 10 6 ) lysed in RIPA buffer. Proteins were resolved by 8% SDS-PAGE and transferred onto nitrocellulose membranes (Thermo Scienti c, USA).
The immunoblots were incubated overnight at 4 °C with the following antibodies: anti-HSP90 (Proteintech), anti-STAU1 (Abcam), anti-KLF16 (Abcam), anti-LIPIN1 (Abcam), anti-UPF1 (Santa Cruz Biotechnology), and anti-pUPF1 (Millipore). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, Beijing, China) at room temperature for 1 h. Densitometric analysis was performed using Image Lab software (Bio-Rad).
The immunoblots were incubated overnight at 4 °C with the following antibodies: anti-HSP90 (Proteintech), anti-STAU1 (Abcam), anti-KLF16 (Abcam), anti-LIPIN1 (Abcam), anti-UPF1 (Santa Cruz Biotechnology), and anti-pUPF1 (Millipore). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, Beijing, China) at room temperature for 1 h. Densitometric analysis was performed using Image Lab software (Bio-Rad).
5
Protein Extraction and Western Blot Analysis
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Cells were collected using a scraper and washed once with cold PBS. Cells were lysed using the RIPA buffer supplemented with protease and phosphatase inhibitors (10 mM Tris-Cl at pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, protease inhibitor, phosphatase inhibitor I and II, 1 mM DTT). Lysates were sonicated, and cell debris was removed through centrifugation. 30~50 µg of total protein was separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane using the Amersham semidry transfer system. The following primary antibodies were used in this study: anti-PKR and anti-peIF2α were purchased from Cell Signaling Technology; anti-pPKR and anti-Stau1 were purchased from Abcam; anti-
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