Western blots: total protein lysates following the indicated treatment were extracted and separated using SDS-PAGE gels according to standard methods. Membranes were probed using the following antibodies: pan Trk clone A7H6R (92991S Cell Signaling Technology), phospho TrkA (Y674/675) clone C50F3 (4621S Cell Signaling Technology), phospho PLCƔ (Y783; 2821L Cell Signaling Technology), phospho MEK1/2 (S217/221) clone 41G9 (9154S Cell Signaling Technology), total MEK1/2 (9122L Cell Signaling Technology), BRAF clone D9T6S (14814S Cell Signaling Technology), BRAF V600E (ab228461 Abcam), phospho p44/42 MAPK (Erk1/2; T202/Y204) clone D13.14.4E (4370S Cell Signaling Technology), total ERK1/2 (9102S Cell Signaling Technology), phospho AKT (S473) clone D9E (4060L Cell Signaling Technology), total AKT (9272S Cell Signaling Technology), pan RAS (BK008, part AESA02 Cytoskeleton), KRAS (F234 Santa Cruz) and β-actin clone 13E5 (4970S Cell Signaling Technology).
β actin clone 13e5
Manufactured by Cell Signaling Technology
Sourced in Japan
β-actin (clone 13E5) is a monoclonal antibody that recognizes the beta-actin protein, a highly conserved cytoskeletal protein found in all eukaryotic cells. The antibody is useful for the detection and quantification of beta-actin as a loading control in western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
Total erk1 2, by Cell Signaling Technology (2 mentions)
Total mek1 2, by Cell Signaling Technology (2 mentions)
Brafv600e, by Abcam (2 mentions)
Pan ras, by Santa Cruz Biotechnology (2 mentions)
Pan trk clone a7h6r, by Cell Signaling Technology (2 mentions)
Phospho trka y674 675 clone c50f3, by Cell Signaling Technology (2 mentions)
Phospho plc y783, by Cell Signaling Technology (2 mentions)
Phospho mek1 2 s217 221 clone 41g9, by Cell Signaling Technology (2 mentions)
Braf clone d9t6s, by Cell Signaling Technology (2 mentions)
Phospho p44 42 mapk erk1 2 t202 y204 clone d13.14.4e, by Cell Signaling Technology (2 mentions)
Phospho akt s473 clone d9e, by Cell Signaling Technology (2 mentions)
Pten 138g6, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Anti flag antibody clone m2, by Merck Group (1 mentions)
Pam3csk4, by Alexis Biochemicals (1 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
10 protocols using β actin clone 13e5
1
Comprehensive Western Blot Analysis
2
Western Blot Analysis of EMT Markers
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Cells were lysed, and proteins were extracted using 1× RIPA buffer; protein concentrations were subsequently determined using the DC™ Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Total proteins were resolved on NuPage 4%–12% Bis-Tris gels (Thermo Fisher Scientific). Proteins were then transferred to membranes using an iBlot® Gel Transfer Device (Thermo Fisher Scientific).
The membranes were incubated with primary antibodies specific for E-cadherin (clone EP700Y; 1:50000 dilution; Abcam, Cambridge, UK), Vimentin (clone H-84; 1:1000; Santa Cruz, Dallas, TX, USA), Fibronectin (clone F1; 1:1000; Abcam), MMP-9 (clone 65-2A4; 1:500; Daiichi Fine Chemical, Toyama, Japan), or β-actin (clone 13E5; 1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. All antibodies were diluted in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% bovine serum albumin. Subsequently, blots were incubated with an horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology; 1:10000) in TBS with 0.1% Tween 20 and 5% nonfat milk for 1 h at room temperature with gentle agitation. Blots were visualized using ImmunoStar® LD (Wako, Tokyo, Japan).
The membranes were incubated with primary antibodies specific for E-cadherin (clone EP700Y; 1:50000 dilution; Abcam, Cambridge, UK), Vimentin (clone H-84; 1:1000; Santa Cruz, Dallas, TX, USA), Fibronectin (clone F1; 1:1000; Abcam), MMP-9 (clone 65-2A4; 1:500; Daiichi Fine Chemical, Toyama, Japan), or β-actin (clone 13E5; 1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. All antibodies were diluted in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% bovine serum albumin. Subsequently, blots were incubated with an horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology; 1:10000) in TBS with 0.1% Tween 20 and 5% nonfat milk for 1 h at room temperature with gentle agitation. Blots were visualized using ImmunoStar® LD (Wako, Tokyo, Japan).
3
CD4+ Cell Signaling Pathway Analysis
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For cell signaling molecules, total CD4+ cells were isolated from secondary lymphoid tissues of mice using MojoSort™ magnetic separation (BioLegend). Cells were activated with anti-CD3/28 ± RA (20 nM) or left unactivated for indicated times. Proteins were extracted from cells with a lysis buffer (1% Triton X-100 and 0.1% SDS in PBS) and probed using antibodies for total Akt (9272), pAkt (T308; 244F9), pS6K (T421/S424; CST #9204), PTEN (138G6), and β-actin (clone 13E5), which were purchased from Cell Signaling Technology.
4
Comprehensive Western Blot Analysis
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Western blots: total protein lysates following the indicated treatment were extracted and separated using SDS-PAGE gels according to standard methods. Membranes were probed using the following antibodies: pan Trk clone A7H6R (92991S Cell Signaling Technology), phospho TrkA (Y674/675) clone C50F3 (4621S Cell Signaling Technology), phospho PLCƔ (Y783; 2821L Cell Signaling Technology), phospho MEK1/2 (S217/221) clone 41G9 (9154S Cell Signaling Technology), total MEK1/2 (9122L Cell Signaling Technology), BRAF clone D9T6S (14814S Cell Signaling Technology), BRAF V600E (ab228461 Abcam), phospho p44/42 MAPK (Erk1/2; T202/Y204) clone D13.14.4E (4370S Cell Signaling Technology), total ERK1/2 (9102S Cell Signaling Technology), phospho AKT (S473) clone D9E (4060L Cell Signaling Technology), total AKT (9272S Cell Signaling Technology), pan RAS (BK008, part AESA02 Cytoskeleton), KRAS (F234 Santa Cruz) and β-actin clone 13E5 (4970S Cell Signaling Technology).
5
Antibody Characterization for Immune Signaling
6
Western Blot Analysis of Apoptosis Markers
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Cells were washed with PBS, then lysed with TN1 lysis buffer (125 mM NaCl, 50 mM Tris, 10 mM EDTA, 1% Triton X-100, 10 mM Na4PO7, 10 mM NaF with 10 mM Na3VO4, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) at 4 ◦C. Lysates were centrifuged (10,000 g, 4 ◦C, 10 min) to remove cell debris, then a Pierce BCA assay (Thermo Scientific, Waltham, MA) was performed to determine protein concentration. Equivalent amounts of denatured and reduced protein were separated by SDS-PAGE and analyzed by Western blot using antibodies against caspse-3 (cat# 9662, lot 19), poly ADP-ribose polymerase (PARP; clone 46D11, cat# 9532, lot 10), and β-actin (clone 13E5, cat# 5125, lot 7), obtained from Cell Signaling (Danvers, MA). The membranes were developed using clarity ECL reagent on a UVP ChemStudio 815 system (Analytik Jena US LLC, Upland CA).
7
Protein Expression Analysis in Human Macrophages
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Nuclear and cytoplasmic protein lysates were isolated from human macrophages using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific) supplemented with Halt protease inhibitor cocktail (Thermo Scientific) according to the manufacturer’s protocol. Samples were separated on 4 to 20% Mini-Protean TGX precast protein gels (Bio-Rad) by SDS-PAGE and transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membranes (EMD Millipore) overnight at 4°C. Membranes were incubated with NF-κB p65 (clone D14E12), TATA binding protein (TBP) (clone D5C9H), or β-actin (clone 13E5) primary antibodies (1:1,000; Cell Signaling), followed by horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:2,000; Cell Signaling). All antibodies were diluted in 1× Tris-buffered saline (TBS)–Tween (TBST) with 5% bovine serum albumin (BSA). Bands were developed by enhanced chemiluminescence and quantified using ImageJ software.
8
Immunoblotting of Phosphorylated CRTC3
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Example 14
Cell lysis and immunoblotting were conducted as described previously (Clark K, et al. (2012) Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages. Proceedings of the National Academy of Sciences of the United States of America 109(42):16986-16991). Briefly, cells were rinsed in ice-cold PBS and extracted in lysis buffer (50 mM Tris.HCl at pH 7.4, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM sodium 3-glycerol 1-phosphate, 1 mM DTT, 1 mM sodium orthovanadate, 1% (vol/vol) Triton X-100 and 1× Complete EDTA-free Protease Inhibitor Cocktail (Roche). Cell extracts were clarified by centrifugation at 14,000×g for 10 min at 4° C., and protein concentrations were determined by using the Bradford assay. To detect proteins in cell lysates, 25 gig of protein extract was separated by SDS/PAGE. After transfer to PVDF membranes, proteins were detected by immunoblotting and visualized by treating the blots with SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific) followed by autoradiography. The following antibodies were used for immunoblotting: β-actin (clone 13E5) was from Cell Signaling; CRTC3 (clone EPR3440) was from Abcam; pSer370 (S253D bleed 2) of CRTC3 was a generous gift of P. Cohen (University of Dundee).
9
Characterization of CatSper Antibodies
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In-house rabbit polyclonal CatSper1 (Ren et al., 2001 (link)), CatSper3 (Qi et al., 2007 (link)), CatSperε (Chung et al., 2017 (link)) antibodies were described previously. Polyclonal CA-IV antibody (M-50) was purchased from Santacruz. Monoclonal antibodies were purchased from BD Biosciences: anti-caveolin1 (clone 2297); EMD Milipore: anti-phosphotyrosine (clon4G10), anti-acetylated tubulin (clone 6-11B-1), anti-HA agarose (clone HA-7); Thermo Scientific: anti-HA magnetic beads; and Cell Signaling Technology: β-actin (clone 13E5) and HA (clone C29F4). HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were from Jackson Immunoresearch. PNA-Alexa 568, WGA-Alexa 555, WGA-Alexa 647, goat anti-mouse IgG (Alexa 488 or 647), and goat anti-rabbit IgG (Alexa 568 or Alexa 647) were from Invitrogen. H89, calyculin A, and calpain inhibitor I were purchased from Calbiochem. ST-Ht31 was from Promega. Calpain inhibitor II and III were from Enzo life science. All other chemicals were from Sigma-Aldrich unless indicated.
10
CD4+ Cell Signaling Pathway Analysis
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