Anti-ALK, anti-phospho-Akt at serine (Ser) 473 (pAkt), anti-Akt, anti-Slug, anti-Snail, and anti-cleaved caspase 3 antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-Sox11 and anti-β-actin antibodies and doxorubicin were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Anti-N-myc, anti-Twist1, and anti-Histone H1 antibodies were from Abcam (Cambridge, MA, USA). Anti-NF-κB/p65, anti-p27kip1, and anti-bax antibodies were from BD Biosciences (San Jose, CA, USA). Anti-bcl-2 and anti-p21waf1 antibodies were from Dako (Glostrup, Denmark). Anti-cyclin A antibody was from Novocastra (Newcastle, UK). Recombinant human tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and hepatocyte growth factor (HGF) were purchased from R&D Systems (Minneapolis, MN, USA).
Anti alk
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-ALK is a laboratory detection reagent produced by Cell Signaling Technology. It is designed to detect the anaplastic lymphoma kinase (ALK) protein in cellular samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti bax, by BD (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti slug, by Cell Signaling Technology (2 mentions)
Anti twist1, by Abcam (2 mentions)
Anti p27kip1, by BD (2 mentions)
Anti met, by Cell Signaling Technology (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti p erk1 2, by Cell Signaling Technology (2 mentions)
Anti phospho alk, by Cell Signaling Technology (2 mentions)
Anti ret, by Cell Signaling Technology (2 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti sox2, by Abcam (2 mentions)
Anti sox7, by Merck Group (2 mentions)
Anti cd44, by Agilent Technologies (2 mentions)
Anti cd133, by Leica Biosystems (2 mentions)
Anti p53, by Agilent Technologies (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
13 protocols using anti alk
1
Antibody Characterization for Cell Signaling
2
Protein Extraction and Analysis Protocol
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Total cell lysates were obtained with NP-40 lysis buffer (150 mM sodium chloride, 1.0% NP-40, 50 mM Tris, pH 8.0), supplemented with protease inhibitor cocktail (cOmplete mini, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Protein samples were denatured with 2-mercaptoethanol at 95 °C for 5 min. The following antibodies were used for detection: anti-pALK Y1604 (Cell Signaling Technology), anti-ALK (Cell Signaling Technology), anti-pSTAT3Y705 (Cell Signaling Technology), anti-STAT3 (Cell Signaling Technology), anti-pERK1/2 (Cell Signaling Technology), anti-ERK1/2 (Cell Signaling Technology), anti-pAkt Y473 (Cell Signaling Technology), anti-AKT, anti-human PARP (Santa Cruz Biotechnology), anti-HDAC8 (H-145;polyclonal; Santa Cruz, Santa Cruz, CA, USA), anti-p-mTOR (Ser2448; Upstate), anti-p-S6K1 (Thr412; Upstate), anti-MET (Cell Signaling Technology), anti-MYCN (Santa Cruz), anti-ac-SMC3 (provided by Prof. K Shirahige, University of Tokyo, Tokyo, Japan) [55 (link)], anti-HSC70 (Santa Cruz), anti-β-actin (clone AC-15; Sigma), anti-actinin (H-2; Santa Cruz) and anti-GAPDH (clone 6C5; Merck).
3
Immunohistochemistry and Western Blot Analysis
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Immunohistochemistry and western blot were carried out according to the previous report.3 (link) For western blot analysis, anti-NeuroD1 (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-Akt (1:500; Cell Signaling Technology), anti-P-Akt (1:500; Cell Signaling Technology) and anti-ALK (1:500; Cell Signaling Technology) antibodies were used.
4
Western Blotting Analysis of Neuroblastoma Cells
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Neuroblastoma cell lines were seeded in a 10-cm dish at a final density of 1.5×106 cells and allowed to attach overnight. The cells were then treated with DMSO or experimental compounds. After 48–72 h, cells were lysed in CHAPS lysis buffer with protease and phosphatase inhibitors (Roche, Basel, Switzerland), and 25 or 50 μg of total lysate were boiled for 5–10 min in CHAPS and sample buffer. Western blotting was performed using the following primary antibodies: anti-ALK (Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-ALK (Cell Signaling Technology), anti-Shc (Cell Signaling Technology), anti-Phospho-Shc (Cell Signaling Technology), anti-PARP (Cell Signaling Technology), anti-cleaved Caspase-3 (Cell Signaling Technology), anti-GAPDH (Fuji Film-Wako Chemicals), and anti-Actin (Merck). Secondary antibodies used were as follows: HRP-linked anti-rabbit IgG (Cell Signaling Technology) and HRP-linked anti-mouse IgG (Cell Signaling Technology). The proteins were visualized using ImageQuant LAS 4000mini with enhanced chemiluminescence reagents (Thermo Fisher Scientific).
5
Protein Isolation and Western Blotting
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Protein isolation and western blotting was performed as previously described5 (link). The following primary antibodies were used: anti-pY1604-ALK (1:500, 3341), anti-ALK (1:750, 3333), anti-pERK1/2 (1:500, 9101S) and anti-ERK1/2 (1:500, 9102), all from Cell Signaling; anti-ETV5 from Abnova (1:500, H00002115-M01). For the loading control, anti-β-actin (Cell Signaling), anti-vinculin (Sigma Aldrich) and anti-GAPDH (Genetex) antibodies were used. Secondary antibodies were used from Cell Signaling. Imaging was done using the Amersham Imager 680 (GE Healthcare).
6
Flow Cytometric Analysis of Receptor Expression
7
Immunoprecipitation and Western Blotting
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All procedures were performed at 4 °C. Cells were lysed in CHAPS lysis buffer [50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10 mM CHAPS, and 1 × Protease Inhibitor cocktail (Roche)]. The lysates (500–2,000 μg protein) and 10 ml conditioned medium were precleared with 20 μl protein A agarose bead (Invitrogen) 50% slurry in 500 μl IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 1 × Protease Inhibitor cocktail) for 30 minutes. The precleared samples were then incubated with anti-HA tag (Roche), anti-ALK (Cell Signaling Technology), and anti-NLRR1 (prepared in-house or purchased from R&D Systems) antibodies for 1 hour, followed by addition of 20 μl protein A agarose bead 50% slurry. Normal rabbit IgG (Santa Cruz Biotechnology) was employed for the negative control. After overnight rotation, the beads were washed six times with 500 μl IP buffer (rotated for 10 minutes twice and inverted four times) and then subjected to western blotting.
8
Antibody and Inhibitor Panel for Cancer Signaling
9
Flow Cytometric Analysis of Receptor Expression
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Cells were washed in PBS and fixed with 2% paraformaldehyde in PBS for 10 min. at 37°C. After washing in PBS, cells were permeabilized in cold 90% methanol for 30 min. on ice. Cells were then washed in 0.5% BSA in PBS and stained with anti-ALK (Cell Signaling Technology, #3633P, 1:400) or anti-RET (Cell Signaling Technology, #3223S, 1:50) for 1 hour at room temperature, followed by a wash in 0.5% BSA in PBS, and staining with anti-rabbit Alexa Fluor 488 secondary antibody (Life Technologies, #A11070, 1:500) for 30 min at room temperature. After a final wash, cells were resuspended in PBS and analyzed on a BD LSR Fortessa flow cytometer.
10
Immunohistochemical Analysis of Oncogenic Markers
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Anti-ALK, Anti-Rb phospho-Ser807/811 (pRb), anti-CD56, anti-synaptophysin (Syn), and anti-vimentin, anti-Slug, anti-cleaved caspase-3, and anti-cleaved poly (ADP-ribose) polymerase 1 (PARP1) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-Sox7, anti-ZEB1, and anti-β-actin antibodies were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Anti-Snail, anti-Nestin, and anti-Sox2 antibodies were from Abcam (Cambridge, MA, USA). Anti-Rb, anti-p27kip1, anti-N-cadherin, anti-BAX, anti-X-linked inhibitor of apoptosis (XIAP), and anti-aldehyde dehydrogenase (ALDH)1 antibodies were from BD Biosciences (San Jose, CA, USA). Anti-Ki-67, anti-p21waf1, anti-cyclin D1, anti-p53, anti-BCL2, and anti-CD44s antibodies were from Dako (Glostrup, Denmark). Anti-cyclin A2, anti-cyclin B1, and anti-CD133 antibodies were from Novocastra (Newcastle, UK), Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Miltenyi Biotechnology (Bergish Gladbach, Germany), respectively. Adriamycin (ADR: Catalog No. #D1515) was purchased from Sigma-Aldrich Chemicals.
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