Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulphate) supplemented with a Protease Inhibitor Cocktail Set III (Merck). Proteins were separated by SDS-PAGE and immunoblot analysis was performed as described elsewhere.8 (link) Horseradish peroxidase-conjugated sheep anti-mouse (NA931V, Cytiva, Marlborough, MA, USA), goat anti-rat (NA935V, Cytiva), and donkey anti-rabbit IgG (NA9340V, Cytiva) served as the secondary antibody for chemiluminescent detection. ImmunoStar LD (Fujifilm Wako Pure Chemical) was used as chemiluminescence reagent and images were taken with the Amersham Imager 600 (Cytiva).
Na9340v
Manufactured by Cytiva
Sourced in United States
The NA9340V is a laboratory equipment designed for liquid handling applications. It features a comprehensive set of functions that facilitate precise and reliable pipetting tasks in various research and analytical settings. The core function of this product is to provide precise and controlled liquid transfer capabilities to support a wide range of laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Amersham imager 600, by Cytiva (1 mentions)
Protease inhibitor cocktail set 3, by Merck Group (1 mentions)
Ab235546, by Abcam (1 mentions)
Nb300 221, by Novus Biologicals (1 mentions)
Ab22552, by Takara Bio (1 mentions)
Ab19027, by Abcam (1 mentions)
Total gsk3β, by Cell Signaling Technology (1 mentions)
Cell signaling lysis buffer, by Cell Signaling Technology (1 mentions)
Pβ catenins33 37 t41, by Cell Signaling Technology (1 mentions)
Novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
P β catenin s675, by Cell Signaling Technology (1 mentions)
Total gsk3α, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Phosstop phosphatase inhibitor, by Roche (1 mentions)
Edta free protease inhibitor cocktail tablet, by Roche (1 mentions)
Western lightning chemiluminescence reagent, by PerkinElmer (1 mentions)
Rpn4201, by Cytiva (1 mentions)
Prism 6, by GraphPad (1 mentions)
7 protocols using na9340v
1
Immunoblot Analysis of Protein Lysates
2
Quantifying Protein Expression via Western Blotting
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Ten microliter aliquots of the HEK control, Clover, and DnaJB8–Clover cell lines were removed from the elution and loaded onto a 4–12% Bis-Tris SDS-PAGE gel for western blotting. Upon running the gel to completion, the gel transferred onto a transfer membrane soaked in Novoblot transfer buffer. Following the transfer, the membrane was soaked in milk blocking buffer for 1 h at room temperature. For immunolabelling, we added 1:2000 dilution of polyclonal anti-GFP (rabbit) (Rockland; 600-401-215; 35460) or anti-DnaJB8 (rabbit) (Abcam; ab235546; GR3229943-2) in milk and incubated the membrane shaking at room temperature for 2 h. For imaging loading standards, we used monoclonal anti-GAPDH (mouse) (Novus Biologicals; NB300-221; 082219). The primary antibody solution was dumped and the membrane washed three times for 10 min each with 1× TBST before adding the polyclonal anti-rabbit IgG peroxidase (Cytiva; NA9340V; 16908235) or polyclonal anti-mouse IgG peroxidase (Cytiva; NA931V; 17089105) at a 1:5000 dilution in milk. The membrane was incubated with a secondary antibody at room temperature for 1 h before removing the antibody solution. The membrane was washed three times in 5-min intervals with 1× TBST and finally one 5-min wash with 1× TBS. The membrane was soaked in 1 mL of Luminol enhancer and peroxide solution for 1 min before imaging.
3
Immunohistochemical Analysis of Bone Cells
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After deparaffinization of sections mounted on slides, endogenous peroxidase was blocked using 3% H2O2 for 30 min at room temperature (RT) and nonspecific blocking with 2% goat serum for 20 min at RT. Bones were assessed by immunohistochemistry for numbers of immature osteoblasts, mature osteoblasts, and osteoclasts using primary antibodies against osterix (Sp7) (Abcam, #ab22552), osteocalcin (OC) (Takara, #M173), and cathepsin K (Abcam, #ab19027), respectively. Primary antibodies diluted in PBST/1%BSA and negative control were applied to the sections overnight at 4°C, and secondary antibody (Amersham, NA9340V) was diluted in PBST/1% BSA and applied for 45 min at RT. Positive staining was detected using DAB (3,3′-diaminobenzidine) Substrate Kit for Peroxidase (Vector, Cat# SK-4100) according to the manufacturer’s protocol, and counterstaining was performed with hematoxylin (Mayer, Sigma, 51 275) for 15 s.
4
Western Blot Analysis of GSK-3 and β-Catenin
5
ELISA-Based Murine IL-1beta Binding Analysis
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Example 6
The binding analysis was carried out using an enzyme-linked immunosorbent assay (ELISA)-based technology. The antigen murine IL-1beta (Sino Biologics, Cat. No. 50101-MNAE) was immobilized at a concentration of 0.5 μg/mL in 25 μL in PBS on a 384 well microtiter plate (Thermo Scientific, Cat. No. 464718). Every of the following steps was followed by a washing routine of 3 times 90 μL PBS, 0.5% BSA, 0.05% Tween with dispense and aspiration: 1) blocking step: saturating unbound surface (1 hour, 2% BSA); 2) anti-IL-1beta antibody in increasing concentrations for 1 hour; 3) detection antibody, dilution=1:2000 (Donkey F(ab)2 anti-rabbit IgG POD, Amersham, NA9340V or sheep IgG anti-mouse IgG POD, Amersham RPN4201). 20-30 min. after adding the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Roche Diagnostics GmbH, Mannheim, Germany, Cat. No 11835033001) the optical density was determined at 370 nm. The EC50 was calculated with a four parameter logistic model using GraphPad Prism 6.0 software.
6
Western Blot Analysis of NF-κB Signaling
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Cell lysates were collected using RIPA buffer (Sigma Aldrich S.r.l., Milan, Italy) adding a protease inhibitor cocktail (Complete Tablets, Roche Diagnostic GmbH, Mannheim, Germany) and sonicated until solubilized. Samples were mixed with 4x Laemmli loading buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and loaded onto 4–20% Tris-Glycine Gels (Bio-rad), and electrophoresis was performed for 50 min at 200 V. The protein was then transferred to a polyvinylidenedifluoride membrane (Thermo Scientific, Rockford, IL, USA) and probed overnight with the primary anti-bodies phospho-NF-kB p65, Ser 536 (Cell Signaling Technology, Danvers, MA, USA) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with HRP-conjugated secondary antibodies (NA9340V and NA931V, against rabbit and mouse primary antibodies, respectively, Amersham Life Science, Milan, Italy).The proteins were detected by Western BrightTM ECL (Advansta, CA, USA), exposed to film and analyzed using a BIORAD Geldoc 2000. The presented data werecalculated after normalization with GAPDH. Densitometrical data obtained from Quantity One software (Bio-Rad) wereapplied for statistical analysis and normalized against the housekeeping GAPDH. The results were expressed as a number-fold vs. untreated cultures, respectively.
7
Western Blot Analysis of GFP Expression
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HEK-293 cells were transiently transfected with GFP1-10 constructs. After 48 hours cells were washed twice with PBS and lysed with M-PER Lysis buffer (Pierce) containing 1x cOmplete Ultra protease inhibitors (Roche). Equal volumes of lysate were separated by SDS-PAGE (4–12 % BisTris NUPAGE gel, Invitrogen) and transferred to PVDF membrane by iBlot (Invitrogen). Immunoblots were processed in the BenchPro blotting system (Invitrogen), using an anti-GFP rabbit primary antibody (G1544, Sigma) and anti-rabbit IgG-HRP secondary antibody (NA9340V, Amersham). Blots were visualized with ECL reagents (Amersham) and images captured by ChemiDoc Imager (BioRad).
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