Cells were lysed in ice-cold NP-40 lysis buffer (Santa Cruz Biotechnology, #sc-24948) with phosphatase and protease inhibitors added, and protein concentrations were measured using the BCA Protein Assay Kit (ThermoFisher, #23227). Lysates were run on 7.5% gradient SDS-polyacrylamide gels and transferred using the methanol-based wet-transfer technique onto 0.45 μm pore-size nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked with 5% phosphoBLOCKER (Cell Biolabs, # AKR-103) and incubated overnight at 4°C for primary antibody binding and with 5% nonfat dry milk reconstituted in Tris-Buffered Saline (TBS) for one hour at room temperature for secondary antibody binding. Blots were developed on standard radiographic film using either Pierce ECL, SuperSignal™ West Pico PLUS, or West Femto Chemiluminescent Substrates (ThermoFisher). All antibodies were obtained from Cell Signaling Technology. Primary antibodies against phosphorylated p44/42 MAPK (ERK1/2)(Thr202/Tyr204) (#9101), phosphorylated STAT1 (Tyr701) (#9167), phosphorylated STAT3 (Tyr705) (#9131), p44/41 MAPK (ERK1/2) (#9102), STAT1(#9172), and STAT3 (#9132) were used at 1:1000 dilutions. Secondary anti-rabbit antibody (#7074) or anti-mouse antibody (#7076) used at 1:5000 dilution for each primary antibody as appropriate.
Phosphoblocker
Manufactured by Cell Biolabs
Sourced in United States
PhosphoBLOCKER is a blocking buffer designed to reduce non-specific binding of phospho-specific antibodies in Western blotting and ELISA applications. It contains a proprietary blend of blocking agents to effectively block non-specific interactions while preserving the detection of phosphorylated proteins.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p pvdf membrane, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Protease and phosphatase inhibitor, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
West femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Pore size nitrocellulose membrane, by Bio-Rad (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
P erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
P ire1α, by Abcam (1 mentions)
Grp78, by Santa Cruz Biotechnology (1 mentions)
Ire1α, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated anti mouse or anti rabbit antibody, by Santa Cruz Biotechnology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti mouse igg, by R&D Systems (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
18 protocols using phosphoblocker
1
Western Blot Analysis of Signaling Proteins
2
Western Blot Analysis of Melanoma Cells
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Melanoma cells were lysed in RIPA buffer containing 50 mmol/l Tris–HCl pH 8.0, 150 mmol/l NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS supplemented with freshly added protease and phosphatase inhibitors (Sigma-Aldrich). Cell lysates were diluted in 2 × Laemmli buffer and protein samples (15 μg) were loaded on standard 7% SDS–polyacrylamide gel. After electrophoresis, the proteins were transferred onto Immobilon-P PVDF membrane (Millipore, Billerica, MA, USA) followed by incubation in a blocking solution: 5% nonfat milk in PBS-Tween 0.05% or 5% phospho-BLOCKER (Cell Biolabs, San Diego, CA, USA) in PBS-Tween 0.05%. Primary antibodies detecting PARP, ATF6, IRE1α, GRP78 and p53 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), p-IRE1α from Abcam (Cambridge, UK), β-actin from Sigma-Aldrich, p-ERK1/2 (Thr202/Tyr204) and ERK1/2 from Cell Signaling Technology (Danvers, MA, USA). Secondary HRP-conjugated anti-mouse or anti-rabbit antibodies (Santa Cruz Biotechnology) and Pierce ECL Western Blotting Substrate (Pierce, Rockford, IL, USA) were used to visualize proteins on the X-ray film (Foton-Bis, Bydgoszcz, Poland) or by using ChemiDoc Imaging System (Biorad). The quantification of the Western blotting data was performed by using ImageJ software.
3
Nuclear Protein Fractionation and Western Blotting
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Western blotting was performed as described previously.40 (link) To isolate nuclear fractions, cells were treated with a hypotonic buffer for 5 min on ice. Proteins were separated by sodium-lauryl-sulfate-polyacrylamide gel electrophoresis and transferred electro-phoretically onto PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% (W/v) phospho-blocker (Cell Biolabs. Inc., San Diego, CA, USA) for 1 h and incubated with primary antibodies such as anti-β-actin (A2066, Sigma, St Louis, MO, USA), anti-phospho-AKT-Ser473 (4060, Cell Signaling, Beverly, MA, USA), anti-AKT (4685, Cell Signaling), anti-cyclin D1 (Nichirei Bioscience), for 1 h at room temperature or overnight at 4 °C. Membranes were then incubated for 1 h at room temperature with the secondary antibody of HRP-conjugated goat anti-rabbit IgG (GE Healthcare, Buckinghamshire, UK) or HRP-conjugated goat anti-mouse IgG (R&D Systems, Minneapolis, MN, USA). The protein bands were visualized with Chemi-Lumi One L Western blotting substrate (NacalaiTesque). Band intensity was measured by densitometry using the Image Lab Software (Bio-Rad).
4
Western Blot Analysis of PARP Cleavage
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After 24 h of irradiation, lysates were generated with a lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.5 M NaCl, 1% Triton X-100). Samples were prepared in a sample buffer and heated to 95 °C for 5 min, run over 10% polyacrylamide gels and separated. The proteins were then transferred to PVDF membranes (Millipore), which were blocked for 3 h in TBST (Tris buffer solution with 0.2% Triton X) containing 5% skim milk and 5% phosphoBLOCKER (Cell Biolabs, San Diego, CA). Next, the membranes were incubated overnight at 4 °C with a primary antibody against poly (ADP-ribose) polymerase (PARP) and cleaved PARP, then washed in TBST three times and incubated with a secondary antibody, anti-rabbit IgG and anti-mouse IgG conjugated with Streptavidin Horseradish Peroxidase Conjugate (GE Healthcare BioSciences, Little Chalfont, UK) for 1 h. Next, the membranes were washed six times in TBST, and luminescence from the Horseradish Peroxidase was detected on X-ray film (Fuji Photo Film Ltd, Tokyo, Japan) with the aid of ECL Western Blotting Detection Reagents (GE Healthcare BioSciences).
5
Quantitative Protein Analysis by Western Blot
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Western blotting was performed using whole cell lysates as described.35 PhosphoBLOCKER (Cell Biolabs, Inc., San Diego, CA) was used for blocking nonspecific binding sites. Antibodies against OLFM4, GRIM19, LGR5, CD133 (Abcam), p‐STAT3 (Ser727), β‐actin, or caspase‐3 (Cell Signaling Technology, Danvers, MA) were used for primary antibody. Immunoreactive proteins were detected by enhanced chemiluminescence and quantified by image analysis.
6
Quantifying DNA Damage Response in Melanoma Cells
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Melanoma cells (1.25×105) were seeded onto a cover glass placed inside a 24-well plate. Cells were left to adhere for 2 h, after which vemurafenib or trametinib was added for 12 h. Next, the culture medium was discarded, and cells were washed with PBS followed by fixation/permeabilization with methanol for 10 min. Cells were incubated in a blocking solution: 5% phosphoBLOCKER (Cell Biolabs, San Diego, CA, USA) in PBS-Tween 0.05% for 1 h. Next, primary antibodies against phospho-H2AX (Ser139, #2577) (Cell Signaling Technology, Danvers, MA, USA) were added for 1 h, and cells were washed with PBS-Tween 0.05% followed by 1 h incubation with secondary, Alexa Fluor555-conjugated (#ab150078) antibodies from Abcam (Cambridge, UK). After washing with PBS-Tween 0.05%, the coverslips were flipped onto a drop of ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA, USA) on glass slides and left to dry overnight. Images were obtained using the Nikon Eclipse TE2000-S inverted microscope (Nikon) with Plan-Apochromat 60×/1.4 Oil DIC N2 objective and Nikon C1 confocal attachment. Nuclear fluorescence was imaged with 408 nm excitation and 432–467 nm emission range, while secondary antibody fluorescence was imaged with 543 nm excitation and 567–642 nm emission range.
7
Protein Immunodetection in Membrane Samples
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Protein extracts were obtained and processed as previously described [50 (link)]. Membranes were blocked with 2.5% phosphoBlocker (Cell Biolabs) in Tris-Buffered Saline (TBS)-Tween 20. For protein immunodetection, the specific primary antibodies were used: Mcl-1, phospho(Ser536)-p65 (p-p65; Cell Signaling Technology) and β-actin (Sigma-Aldrich). Anti-rabbit and anti-mouse horseradish peroxidase-labeled IgG (Sigma-Aldrich) were used as secondary antibodies. Chemiluminescence was detected with ECL substrate (Pierce Biotechnology) on a mini-LAS4000 Fujifilm device (GE Healthcare). Signal was quantified with Image Gauge densitometric software (Fujifilm) and referred to the respective untreated control.
8
Signaling Pathways in Melanoma Cells
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Melanoma cells were lysed in RIPA buffer containing 50 mmol/l Tris-HCl pH 8.0, 150 mmol/l NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS supplemented with freshly added protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentration was determined by Bradford assay. Cell lysates were diluted in 2x Laemmli buffer and protein samples (15 μg each) were loaded on 7% SDS-polyacrylamide gel. The proteins were transferred onto an Immobilon-P PVDF membrane (Millipore, Billerica, MA, USA). The membrane was incubated in a blocking solution: 5% nonfat milk in PBS-Tween 0.05% or 5% phosphoBLOCKER (Cell Biolabs, San Diego, CA, USA) in PBS-Tween 0.05%. Primary antibodies detecting PARP and total β-catenin were from Santa Cruz Biotechnology, active β-catenin (dephosphorylated on Ser37 and Thr41) from Millipore (Temecula, CA, USA), p-p65 (Ser536), p65, p-ERK1/2 (Thr202/Tyr204), ERK1/2 from Cell Signaling Technology (Danvers, MA, USA). Immunodetection of β-actin (Sigma-Aldrich) was used as a loading control.
9
Immunoblotting Analysis of Cellular Signaling
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Isolated cells with or without stimulation were washed with phosphate-buffered saline (PBS) and lysed with NP-40 buffer (NaCl 150 mM, Tris 50 mM, Nonidet P-40 1%, sodium pyrophosphate 4 mM) or RIPA buffer (NaCl 150 mM, Tris 50 mM, Nonidet P-40 1%, sodium deoxycholate 0.5%, SDS 0.1%) supplemented with a Protease Inhibitors cocktail (Sigma) plus 1mM sodium vanadate, 1mM sodium fluoride, 1 mM PMSF. Lysed samples were spun down at 10,000 g to remove debris and boiled at 100°C for 10 min, with Tris-Glycine buffer (SDS 2%, Bromophenol Blue 0.01%) containing 0.1 M DTT. Aliquots were separated via SDS-PAGE. transferred onto PVDF membrane using the Trans-Blot semi-dry transfer system (Bio-Rad). Samples probed for phosphorylation were specially blocked with 5% phosphoBLOCKER (Cell Biolabs) before probing with the following antibodies: anti-SHP-1 (Invitrogen), anti-AKT, or anti-S473-pAKT (Cell Signaling). Blots were re-probed with anti-β-actin HRP (Sigma) to control for loading.
10
Western Blot for Protein Expression
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To detect the protein expression (except for pATM), samples were separated on 12% Tris-glycine gel and transferred on PVDF membranes. After blocking for 1 h with 0.2% I-Block (Applied Biosystems) in TBST (TBS with 0.5% Tween20), membranes were incubated with indicated antibody overnight. The antibody signal was detected with HRP-conjugated secondary antibodies (Promega) using the ECL reagents (GE healthcare) and exposed to X-ray films (SuperRX, Fujifilm).
For detection of pATM, samples were separated on NuPAGE 3–8% Tris-Acetate protein gel (Invitrogen) and transferred on PVDF membrane with 25 V for 15 h. After blocking for 1 h with 5% PhosphoBLOCKER (Cell Biolabs, AKR-104) in TBST, membranes were incubated with indicated antibody for 2 h at room temperature. All subsequent steps were performed as described above.
For detection of pATM, samples were separated on NuPAGE 3–8% Tris-Acetate protein gel (Invitrogen) and transferred on PVDF membrane with 25 V for 15 h. After blocking for 1 h with 5% PhosphoBLOCKER (Cell Biolabs, AKR-104) in TBST, membranes were incubated with indicated antibody for 2 h at room temperature. All subsequent steps were performed as described above.
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