Cells were washed two times with ice-cold PBS and lysed on ice in 50–60μl of 1X Laemmli sample buffer (60 mM Tris-Hcl, pH 6.8, 10% glycerol, 2% SDS). Protein determination was performed using the Biorad DC protein assay and 0.01% bromophenol and 5% β-mercaptoethanol were added. Western blotting was performed as described [48 (link)]. Briefly, 20 μg protein was loaded per lane on Bio-Rad TGX 4–15% or AnyKD Criterion gels. Following transfer to nitrocellulose, proteins were detected using the following antibodies: CAV1 (D46G3, Cell Signaling), CAV2 (610685, BD Transduction Laboratories), CAV3 (610421, BD Transduction Laboratories), Cavin-1/PTRF (ab48824, Abcam), Cavin-2/SDPR (ab113876, Abcam), Cavin-3/PRKCDBP (16250-1-AP, Proteintech), Tropomyosin/TPM1 (3910, Cell Signaling), SM22α/TAGLN (ab 14106, Abcam), HSP90 (610418, BD Transduction Laboratories) and GAPDH (MAB374, Millipore), all at recommended dilutions. Secondary HRP-conjugated anti mouse or anti rabbit antibodies (Cell Signaling) were then used for detection by enhanced chemiluminescence (Pierce West Femto). An Odyssey Fc Imager (LI-COR Biosciences) was used for image capture and Image Studio 3.1 was used for analysis.
Hrp conjugated anti rabbit or anti mouse antibody
Manufactured by Cell Signaling Technology
Sourced in United States
HRP-conjugated anti-rabbit or anti-mouse antibodies are secondary antibodies that are conjugated with horseradish peroxidase (HRP) enzyme. These antibodies are designed to detect and bind to primary antibodies raised in rabbit or mouse, respectively. The HRP enzyme can be used to catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and visualization of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Ccd camera, by Fujifilm (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Odyssey fc imager, by LI COR (2 mentions)
Sds page, by Fujifilm (2 mentions)
Anti erk 137e5, by Cell Signaling Technology (2 mentions)
Anti α tubulin b 5 1 2, by Santa Cruz Biotechnology (2 mentions)
Ab14106, by Abcam (1 mentions)
Ab48824, by Abcam (1 mentions)
Mab374, by Merck Group (1 mentions)
Hsp90, by BD (1 mentions)
Cav 2, by BD (1 mentions)
Tgx 4 15 criterion gels, by Bio-Rad (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Anti phospho nf κb p65 s536 and anti nf κb p65 93h1 and d14e12, by Cell Signaling Technology (1 mentions)
Anti iκbα l35a5, by Cell Signaling Technology (1 mentions)
Anti phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Anti fak, by Cell Signaling Technology (1 mentions)
Anti nf κb p65 d14e12, by Cell Signaling Technology (1 mentions)
Anti phospho nf κb p65 ser536 93h1, by Cell Signaling Technology (1 mentions)
14 protocols using hrp conjugated anti rabbit or anti mouse antibody
1
Western Blot Analysis of Caveolins
2
Western Blot Analysis of Caveolar Proteins
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Cells were washed using ice-cold PBS and lysed in 70 μl of 1X Laemmli sample buffer (60 mM Tris-Hcl, pH 6.8, 10% glycerol, 2% SDS). Protein determination was performed using the Biorad DC protein assay and 0.01% bromophenol and 5% β-mercaptoethanol were added. 20μg protein was loaded per lane on TGX 4–15% Criterion gels (Bio-Rad, no. 567-1084). Following 1D separation and transfer to nitrocellulose, proteins were detected using the following antibodies: CAV1 (Cell Signaling, cat. D46G3), CAV2 (BD Transduction Laboratories, 610685), CAV3 (BD Transduction Laboratories 610421), CAVIN11 (Abcam ab48824), CAVIN2 (Abcam, ab113876), CAVIN3 (Proteintech, 16250-1-AP), MKL1/MRTF-A (Cell Signaling Technology, #14760), CNN1 (Abcam, ab46794), HSP90 (BD Transduction Laboratories, 610418). Primary antibodies were used at dilutions recommended by the vendors. Secondary HRP-conjugated anti mouse or anti rabbit antibodies (Cell Signaling) were used for detection with SuperSignal West Pico Chemiluminescent Substrate (Pierce, cat34080) in an Odyssey Fc Imager (LI-COR Biosciences). Image Studio 3.1 was used for image capture and analysis.
3
Western Blot Analysis of Cell Lysates
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Cells (5 × 106) were washed once with PBS (without calcium) and sonicated in cold buffer (20 mM Tris, 2.5 mM Sodium Pyrophosphate, 150 mM NaCl, 1 mM Disodium β-Glycerophosphate Pentahydrate, 1 mM ethylenediaminetetraacetic acid, 1 mM 14-bis[(acetyloxy)methyl] ester, 1% Triton-X) for 5 min. The lysates were clarified by brief centrifugation (10,000 × g), and the supernatant was used for analysis. After measuring protein concentrations with a protein assay kit (Bio Rad, Richmond, CA), 20 μg protein of each sample was mixed with an equal volume of SDS-loading buffer (100 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, 2% β-mercaptoethanol), boiled for 5 min, and subjected to SDS-PAGE (Wako, Japan). The separated proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA) that was incubated with 1:1,000-diluted primary antibody and then with HRP-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling Technology) as recommended by the manufacturers. Primary antibody binding was detected using a West Pico chemiluminescent substrate kit (Pierce, Rockford, IL), and images were captured by a CCD camera (Fuji Film, Tokyo, Japan).
4
Western Blot Analysis of Cellular Signaling
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Cells (1 × 105) were washed once with PBS (without calcium), lysed in 100 μl of SDS-loading buffer (100 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, 2% ß-mercaptoethanol) and sonicated for 5 min. The samples were boiled for 5 min and then subjected to SDS-PAGE. The separated proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA), which was then incubated with 1: 1,000-diluted primary antibody and then with HRP-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling Technology) as recommended by the manufacturers. Primary antibody binding was detected using a Clarity Western ECL substrate (Bio Rad, Hercules, CA), and images were captured with a CCD camera (Fuji Film, Tokyo, Japan). The following primary antibodies were used: anti-phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204) (20G11, Cell Signaling Technologies, Danvers, MA, USA), anti-ERK (137E5, Cell Signaling), anti-phospho-Akt (T308) and anti-Akt pan (C31E5E and C67E7, Cell Signaling), anti-phospho-NF-κB p65 (S536) and anti-NF-κB p65 (93H1 and D14E12, Cell Signaling), anti-IκBα (L35A5, Cell Signaling), anti-pIκBα (Ser32, 14D4, Cell Signaling) and anti-α-tubulin (B-5-1-2, Santa Cruz Biotechnology, Dallas, TX, USA).
5
Western Blot Analysis of Cell Signaling Pathways
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Cells (1×105) were washed once with PBS, lysed in 100 μl SDS loading buffer (100 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, and 2% β-mercaptoethanol) and sonicated for 5 min. The samples were boiled for 5 min and subjected to SDS-PAGE, and the separated proteins were transferred onto a PVDF membrane (Merck Millipore) that had been incubated with 1:1,000-diluted primary antibody and then with HRP-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling), as recommended by the manufacturers. Primary antibody binding was detected using a Clarity™ Western ECL substrate (Bio Rad, Hercules, CA, USA), and images were captured by a CCD camera (Fuji Film, Tokyo, Japan). The following primary antibodies were used: anti-pERK (20G11, Cell Signaling), anti-ERK (137E5, Cell Signaling), anti-phospho-Akt (Thr308) (C31E5E, Cell Signaling), anti-Akt pan (C67E7, Cell Signaling), anti-phospho-FAK (Tyr925) (Cell Signaling), anti-FAK (Cell Signaling), anti-phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling), anti-NF-κB p65 (D14E12, Cell Signaling), and anti-α-tubulin (B-5-1-2, Santa Cruz Biotechnology, Dallas, TX, USA).
6
Western Blot Protein Analysis
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Cells were washed once with PBS(-), suspended in SDSloading buffer (100 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, 2% βmercaptoethanol), sonicated for 5 min, and boiled for 5 min, and then subjected to SDS-PAGE (Wako, Japan). The separated proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA), and then blocked with 5% BSA in TTBS for 60 min at room temperature. The membrane was then incubated with 1:1000-diluted primary antibody and then with HRP-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling Technology) as recommended by the manufacturers. Primary antibody binding was detected using a Clarity Western ECL Substrate (Bio-Rad, Richmond, CA), and images were captured by a CCD camera (Fuji Film, Tokyo, Japan).
7
Immunoblot Analysis of Cellular Proteins
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Cell sample harvesting and immunoblot analysis were performed as presented previously [50 (link)]. To detect proteins, the primary antibodies β-actin (sc-47778, Santa Cruz Biotechnology, USA), vinculin (Sigma-Aldrich), HIF-1α (GTX127309, Genetex, USA), HIF-2α (NB100–122, Novus Biologicals, USA or GTX30114, Genetex, USA), p70 s6 kinase and phospho-p70 s6 kinase (Thr 389) (9202 and 9205 respectively, both from Cell Signaling, USA), and MF20 (a hybridoma producing both antibody detecting α and β MHC isoforms; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) were used in 5% non-fat milk/TBS-T at 4°C overnight. Corresponding secondary HRP-conjugated anti-rabbit or anti-mouse antibodies (in 5% non-fat milk/TBS-T, 1 h, RT; Cell Signaling, USA) were employed. Immunoreactive bands were detected using ECL detection reagent kit (Pierce, USA) and exposed to radiographic film (AGFA, Belgium). The adjustment of brightness and contrast was applied to the whole image.
8
Western Blot Analysis of Total Cellular Proteins
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Total cell protein was extracted using a RIPA lysis buffer and centrifuged at 4°C. Protein concentration was determined by a BCA kit (Tiangen, Beijing, China) and separated using 10% SDS-PAGE electrophoresis. The separated proteins were transferred to 0.45 μm nitrocellulose membranes (Millipore, Billerica, MA, United States). The membranes were blocked with 5% BSA and then incubated overnight at 4°C with primary antibodies, which were listed in Supplementary Table S1 . The HRP-conjugated anti-rabbit or anti-mouse antibodies (1/5,000, Cell Signaling Technology, Beverly, MA, United States) was incubated at room temperature, after being rinsed with the PBST solution three times. The membranes were visualized by a high-sig ECL Western blotting substrate (Tanon, Shanghai, China) and detected by an automatic chemiluminescence image analysis system (Tanon, Shanghai, China).
9
Protein Extraction and Western Blotting
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Whole-cell lysates were prepared using RIPA lysis buffer containing 20 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 10% glycerol, 1% Nonidet P-40, and Complete EDTA-free protease inhibitor cocktail (Roche). Nuclear extracts were obtained using a high salt extraction buffer [20 mmol/L HEPES (pH 7.9), 0.32 mol/L NaCl, 1 mmol/L EDTA, and 1 mmol/L EGTA) supplemented with 1 mmol/L DTT and protease inhibitor cocktail. Immunoblotting was performed with primary antibodies (Supplementary Table S2) and detected with either HRP-conjugated anti-rabbit or anti-mouse antibodies (Cell Signaling Technology). Protein bands were visualized by applying ECL Prime reagent (GE Healthcare) using the ChemiDoc Touch Imaging System (Bio-Rad).
10
Western Blot Analysis of Neural Markers
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Brain tissues were homogenized in lysis buffer (150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, 1% Triton X-100, 0.5% SDS) with protease inhibitor cocktail and phosphatase inhibitor (Sigma). Equivalent amounts of protein were analyzed by 4–20% Tris-Glycine gel electrophoresis (Invitrogen). Proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies. Visualization was enabled using enhanced chemiluminescence (GE Healthcare Pharmacia). The following primary antibodies (dilutions) were used: anti-phosphoCREB (1:1000, Cell Signaling, #9198), anti-CREB (1:1000, Cell Signaling, #9197), anti-phosphoERK (1:1000, Cell Signaling, #9106), anti-ERK (1:1000, Cell Signaling, #9102), anti-phospho-CaMKII (1:1000, Cell Signaling, #12716), anti-CaMKII (1:1000, Cell Signaling, #4436), anti-PSD95 (1:1000, Cell Signaling, #36233), anti-BDNF (1:1000, Sigma, #AB1534SP), Iba-1 (1:1000, Santa Cruz Biotechnology, sc-32725), CD68 (1:1000, Bio-Rad, MCA341GA), Dectin-1 (1:1000, Invitrogen, PA5-34382), CD11b (1:1000, Bio-Rad, MCA711), and β-actin (1:5000, Cell Signaling, #3700). Secondary antibodies were HRP-conjugated anti-rabbit or anti-mouse antibodies (1:1000, Cell Signaling, #7074 and #7076).
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