Muscle HSP was assessed by Western blotting for muscle HSP25 and HSP72 content using primary polyclonal antibodies specific to muscle HSP25 (ADI-SPP-715, Enzo, Farmingdale, USA) and muscle HSP72 (ADI−SPA−812, Enzo, Farmingdale, USA), diluted 1:1000 in TBST and 2% milk, with secondary antibody diluted 1:20,000 in 3% milk. The overall Western blotting protocol has been previously described [45 (link)].
Adi spa 812
Manufactured by Enzo Life Sciences
Sourced in United States
The ADI-SPA-812 is a piece of laboratory equipment used for sample preparation and analysis. It is designed to perform solid-phase extractions (SPE) to isolate and purify target analytes from complex matrices. The core function of the ADI-SPA-812 is to automate the SPE process, allowing for increased efficiency and reproducibility in sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Adi spp 715, by Enzo Life Sciences (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Anti actin, by Merck Group (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
A2668, by Merck Group (1 mentions)
Ecl chemiluminescent detection kit, by Thermo Fisher Scientific (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Protein a agarose beads, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti flag, by Merck Group (1 mentions)
Gapdh nb 600 502, by Novus Biologicals (1 mentions)
F7425, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Dapi h 1200, by Vector Laboratories (1 mentions)
4 protocols using adi spa 812
1
Quantifying Muscle HSP Expression
2
Western Blot Analysis of Hsp70
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Striatal and midbrain samples were suspended in RIPA buffer (Millipore, 20-188) containing protease inhibitor (Roche Diagnostics), homogenized on ice, and centrifuged at 13,000 × g for 10 min. Protein concentration of each lysate was determined by BCA. Twenty microgram proteins was loaded on a denaturing 4–12% Bis- Tris gradient gel (Nupage, Novex Gel, Life tech) and run according to the manufacturer's instructions. Subsequently, separated protein was transferred to polyvinylidene difluoride (PVDF) membrane, blocked in 5% non-fat milk in TBS-T, and immunoblotted for Hsp70 (1:5000, rabbit, ADI-SPA-812, Enzo). Anti-actin (1:10000, rabbit polyclonal, A2668, Sigma-Aldrich) was used as a loading control. All membranes were then incubated with a secondary antibody conjugated to HRP for 1 h at room temperature. Immunoreactivity was visualized using an ECL chemiluminescent detection Kit (Thermo Fisher Sci) and images were acquired with a CCD imaging system (LAS-4000, Fujifilm, Japan).
3
Immunoprecipitation of Hsp70 Protein
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Untreated or nigericin-treated cells (50.106) were lysed in 1 mL lysis buffer (25 mM Hepes (pH8), 150 mM NaCl, 0.5% Triton X-100, 5 mM EDTA, 10% glycerol, 1 mM NaVO4, 20 mM NaF and CPIM) for 30 min on ice. After centrifugation at 14,000 × g at 4 °C for 30 min, supernatants were precleared during 2 h at 4 °C in the presence of 30 μL of mixed Sepharose 6B (6B100, Sigma-Aldrich) and protein G (17-0618-01, Amersham, GE Healthcare). After centrifugation at 1000 g for 3 min the supernatant was incubated with 1 μg/mL of anti-HSP70 antibody (ADI-SPA-812, Enzo life sciences) and 40 μL of mixed Sepharose at 4 °C for 20 h. The precipitates were washed four times in lysis buffer, eluted in loading buffer and analyzed by immunoblotting.
4
Western Blot Antibody Panel for Protein Analysis
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For Western Blot analyses the following antibodies were used: anti-β-actin-HRP (ab8224, 1:10000) (Abcam), rabbit-anti-FLAG (F7425, 1:2000), and mouse-anti-FLAG M2 (F1804, 1:2000) (Sigma), mouse-anti-HSP70/72 (ADI-SPA-810) and rabbit-anti-HSP40 (ADI-SPA-400-D) (Enzo Life Science). Additional primary antibodies were used against Akt (C6E7; Cell Signaling Technology), ERK1/2 (9102, Signaling Technology), GAPDH (NB-600-502; Novus Biologicals), HSP90 (H114, sc7947; Santa Cruz Biotechnology), CHIP (PA5-32046, Thermo Scientific, Pierce), HA (MMS-101R, Covance, Biolegend), HSP25 (ADI-SPA-801-488; Enzo Life Science), and HSP70 (ADI-SPA-812; Enzo Life Science). HRP conjugated secondary antibodies were from Bio-Rad (170-6515, 170-6516). For immunofluorescence the following primary antibodies were used: rabbit-anti-FLAG (F7425, 1:200), mouse-anti-FLAG M2 (F1804, 1:200) (Sigma), AlexaFluor conjugated secondary antibodies were from Life Technologies (A11072, 1:300). Mounting medium with DAPI (H-1200, 1:500) was from Vector Labs. For immunoprecipitation, pull down was performed with protein A–agarose beads (Santa Cruz Biotechnology) and the anti-AR antibody (PG21; Millipore).
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