Total protein levels of lysates from HEK293T cells were quantified using the BCA Protein Assay kit (Novagen) as per manufacturer's protocol. 20 µg of total lysates were denatured at 95°C in 4X loading buffer (125 mM TrisHCl pH 6.8, 6% SDS, 4 M urea, 4 mM EDTA, 30% glycerol, 4% β-mercaptoethanol and Bromophenol blue) and loaded on NuPAGE 4-12% Bis-Tris Gel (Life Technologies). Proteins were transferred on PVDF membranes using the transfer apparatus from Life Technologies or Trans-Blot Turbo Transfer System from Biorad following manufacturer's protocol. Membranes were stained for 30 minutes in TBS, 0.1% Tween, 0.4% PFA, before blocking (1 hr) in TBS, 0.1% Tween, 5% non fat milk. Incubations with primary antibodies were performed overnight at 4°C. Incubations with secondary anti-mouse or -rabbit horseradish peroxidase conjugated antibodies were carried out for 1 hour at room temperature. Protein bands were detected using chemiluminescence substrate (ECL from Life Technologies). Similarly 0.25 µg or 2–5 µg of human recombinant HTT N548 proteins were loaded on NuPAGE 4–12% Bis-Tris Gel (Life Technologies) for Western blotting analysis and for fast coomassie-G250 staining respectively.
Nupage 4 12 bis tris gel
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, France, Canada, Switzerland, Japan
The NuPAGE 4–12% Bis-Tris gel is a pre-cast polyacrylamide gel used for protein separation and analysis. It provides a consistent and reliable platform for electrophoretic separation of proteins in a Bis-Tris buffered system.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (92 mentions)
Ripa buffer, by Thermo Fisher Scientific (62 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (60 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (48 mentions)
Nitrocellulose membrane, by Bio-Rad (47 mentions)
Pvdf membrane, by Merck Group (46 mentions)
Protease inhibitor cocktail, by Roche (43 mentions)
Protease inhibitor cocktail, by Merck Group (38 mentions)
Ripa buffer, by Merck Group (38 mentions)
β actin, by Merck Group (36 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (35 mentions)
Complete protease inhibitor cocktail, by Roche (30 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (30 mentions)
Odyssey blocking buffer, by LI COR (29 mentions)
Pvdf membrane, by Thermo Fisher Scientific (29 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (29 mentions)
Bradford assay, by Bio-Rad (27 mentions)
Nitrocellulose membrane, by GE Healthcare (27 mentions)
Ecl plus western blotting substrate, by Thermo Fisher Scientific (23 mentions)
Image lab 6, by Bio-Rad (23 mentions)
1 261 protocols using nupage 4 12 bis tris gel
1
Quantification and Western Blot Analysis of Proteins
2
Quantitative Proteomics in Cell and Fly Models
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Total protein levels of lysates obtained from HEK293T cells and Drosophila HD model, the latter produced as in ref. 9 (link), were quantified using the Pierce BCA Protein Assay kit (Thermo Scientific) according to manufacturer’s protocol. 20 µg of total lysates were denatured at 95 °C in 4x loading buffer (125 mM TrisHCl pH 6.8, 6% SDS, 4 M urea, 4 mM EDTA, 30% glycerol, 4% β-mercaptoethanol and Bromophenol blue) and loaded on NuPAGE 4–12% Bis-Tris Gel or WedgeWell 6% Tris-Glycine Gel (Life Technologies). Proteins were transferred on PVDF membranes using the transfer apparatus from Life Technologies or Trans-Blot Turbo Transfer System from Biorad following manufacturer’s protocol. Membranes were stained for 30 minutes in TBS, 0.1% Tween, 0.4% PFA, before 1 hour blocking in TBS, 0.1% Tween, 5% not fat milk. Incubations with primary antibodies were performed overnight at 4 °C. Incubations with secondary anti-mouse- or rabbit horseradish peroxidase conjugated antibodies were carried out for 1 hour at room temperature. Protein bands were revealed using chemiluminescence substrate (ECL from Life Technologies) and images were acquired using a Chemidoc imager (Biorad). Similarly, synthetic huntingtin exon 1 proteins were loaded on NuPAGE 4–12% Bis-Tris Gel (Life Technologies) for Western blotting analysis.
3
Visualizing Viral Capsid Proteins
4
SDS-PAGE Analysis of Protein Digests
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SDS-PAGE analysis was conducted on the various digesta samples as well as the initial undigested matrices, after dilution of all samples to 0.5% protein in milliQ water and then further diluted 1:2 in 0.1 mol/L Na-phosphate buffer, pH 7.0. The samples were then mixed 1:1 with 2 × Laemmli sample buffer (20 μL) containing DTT (350 mmol/L) and placed on a heat block (80 °C, 4 min). Protein standards (10 μL, Precision Plus Protein™ Dual Xtra #1610377, BIO-RAD) and the test samples (20 μL) were loaded onto a NuPAGE™ 4–12% Bis-Tris gel (10 well, 1.0 mm, Invitrogen™). Dithiothreitol 99% (DTT, 457779, Sigma-Aldrich), 2x Laemmli Sample Buffer (BIO-RAD, #1610737), NuPAGE™ 4–12% Bis-Tris gel 10 well 1.0 mm (Invitrogen™), NuPAGE™ MOPS SDS Running Buffer (20x, Invitrogen™), Precision Plus Protein™ Dual Xtra (BIO-RAD, #1610377), SimplyBlue™ SafeStain (Invitrogen™). The electrophoresis (40 min, 200V, 120 mA [240 mA for 2 gels], 25W) was performed using NuPAGE™ MES SDS running buffer. The gel was rinsed two times in water, followed by staining with Invitrogen™ SimplyBlue™ SafeStain (60 min, 22 °C). The gel was de-stained in water (100 mL, 1 hour). A NaCl solution (20% w/v, 20 mL) was added to the de-staining water and the gel was incubated overnight. Gel Imaging were performed on a Gel Doc™ EZ System (BIO-RAD) using Image Lab™ software.
5
Fibrinogen Proteolysis by CatroxMP-II
6
Protein Separation and Detection by SDS-PAGE
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Purified proteins were separated by SDS–PAGE (NuPAGE gel 4–12% Bis-Tris, Invitrogen) and visualized either by Coomassie staining or SYPRO Ruby Protein Gel Stain (Invitrogen) using a Typhoon 9400 scanner (Amersham Biosciences) with λex = 457 nm and λem = 610 nm. Alternatively, proteins were separated by SDS–PAGE on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and blotted onto polyvinylidene difluoride (PVDF) membranes (Pierce). Proteins were revealed using the following antibodies at the indicated dilutions: i) polyclonal anti-MPK10 antibody, generated by rabbit immunization using recombinant MPK10 protein produced in E. coli transformed with pGEX-Strep3-MPK10 plasmid (Eurogentec), 1∶10,000; ii) anti-phospho-tyrosine antibody 4G10 Platinum from Millipore, 1∶1,000; and iii) secondary goat anti-rabbit-HRP and anti-mouse-HRP antibodies from Thermo Scientific, 1∶20,000. The visualization was performed on X-ray film (Roche) at various exposure times.
7
Western Blot Analysis of Transduced Cells
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Western blotting was carried out as described [9 (link),68 (link)]. Transduced cells were lysed on the fourth day of post-lentiviral infection. The total protein concentrations were determined using the ProteinAssay Dye Bio-Rad Kit (Bio-Rad Laboratories, Inc. Hercules, CA, USA), according to the manufacturer’s recommendations. Electrophoresis and protein blotting was performed using NuPageTM 4–12%Bis-Tris Gel, 1.5 mm * 15 w (Invitrogen, Waltham, MA, USA), and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Blots were probed with the indicated antibodies. In all blots, proteins were visualized by enhanced chemiluminescence (WesternBrightTM ECL, Advansta, San Jose, CA, USA).
8
SDS-PAGE and Western Blot Analysis
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Proteins were mixed with 5 × Laemmli buffer and incubated at 100 °C for 10 minutes. Samples were immediately transferred in the ice afterwards. All samples and prestained protein ladder (10-250 kDa, Thermo ScientificTM) were loaded into the columns of NuPAGETM 4-12% Bis-Tris Gel (Invitrogen) placed in the Invitrogen tank containing 1× NuPAGE MOPS SDS running buffer (Novex). The gel was run at 100v for 75 minutes and transferred to a PVDF (Polyvinylidene difluoride) membrane (Millipore) using NuPAGE transfer buffer (Novex) with methanol and antioxidant (Invitrogen) at 35v for 60 minutes. The membrane was blocked with 5% milk-TBST at RT for 1 hour and incubated overnight with TRIB1 (Millipore), and HSP90 (Abcam) diluted in 5% milk-TBST (1:1000 and 1:5000 respectively) at 4 °C. The membrane was then washed with 0.1 v/v TBST for 5 minutes 3 times and incubated with Polyclonal Goat anti-Rabbit Immunoglobulin/HRP, and Polyclonal Rabbit anti-Rat Immunoglobulin/HRP (Dako) diluted in 5% milk-TBST (1:2500 and 1:5000 respectively) at RT for 1 hour. The membrane was then washed with TBST 3 times for 5 minutes, incubated with ECL, and imaged with Bio-Rad imager.
9
Western Blot Analysis of Lpxn and Paxillin
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Splenocytes or the RAW264.7 cell line were resuspended in RIPA lysis buffer supplemented with Protease and Phosphatase Inhibitor (Thermofisher Scientific). Five or 30 μg of proteins were separated on a NuPAGETM 4–12% Bis-Tris Gel (Invitrogen) and transferred to a PVDF membrane. Primary antibodies against Lpxn (provided by HO), paxillin family protein (BD Biosciences) or β-actin (Cell Signaling) were incubated overnight at 4°C. Secondary antibodies, anti-rabbit IgG and anti-mouse IgG1, respectively (Jackson Immuno Research and Southern Biotech) conjugated to HRP were incubated 2 h at room temperature. Proteins were detected using Pierce ECL (Thermofisher Scientific) and signal was quantified by ChemiDoc™ Touch Gel Imaging System (BIO RAD). Band intensity was measured with ImageJ, background was subtracted, intensities were normalized to β-actin then to the WT control group.
10
Antibodies and Reagents for FAK, p53, and NF2 Analysis
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Polyclonal antibody to FAK, and monoclonal antibodies to phosphotyrosine (PY99), p53, PARP, and NF2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal mouse antibodies to MDM2 and p21, and polyclonal antibody to phospho-FAK (Y397) were from Zymed Laboratories (Invitrogen Life Technologies, Carlsbad, CA, USA). Polyclonal antibodies to phospho-p53 (Ser15) and phospho-NF2 (Ser518) were from Cell Signaling Technology (Beverly, MA, USA). Phospho-FAK (Y861) was obtained from Biosource (Invitrogen Life Technologies). We obtained monoclonal mouse antibodies to cyclin A from Novocastra (Newcastle upon Tyne, UK), and β-actin and GAPDH from Sigma-Aldrich (St Louis, MO, USA).
Mouse anti-phosphotyrosine (PY)-sepharose 4B, Protein A- and Protein G-sepharose beads were from Zymed Laboratories. NOVEX Coomassie colloidal blue stain kit, NuPAGE TM 4–12% Bis-Tris Gel, Lipofectamine and Plus reagent were obtained from Invitrogen Life Technologies. Phenyl phosphate and polybrene were from Sigma. Nutlin-3 and PF562271 were obtained from LC Labs (Woburn, MA, USA) and Symansis (Auckland, NZ, USA), respectively. Lentiviral NF2 shRNAs were from Sigma. NF2 shRNA1: 5′-CCGGCGGGCTTTGTTTCCTTCTTTACTCGAGTAAAGAAGGAAACAAAGCCCGTTTTTG-3′ NF2 shRNA2: 5′-CCGGGCTTCGTGTTAATAAGCTGATCTCGAGATCAGCTTATTAACACGAAGCTTTTTG-3′.
Mouse anti-phosphotyrosine (PY)-sepharose 4B, Protein A- and Protein G-sepharose beads were from Zymed Laboratories. NOVEX Coomassie colloidal blue stain kit, NuPAGE TM 4–12% Bis-Tris Gel, Lipofectamine and Plus reagent were obtained from Invitrogen Life Technologies. Phenyl phosphate and polybrene were from Sigma. Nutlin-3 and PF562271 were obtained from LC Labs (Woburn, MA, USA) and Symansis (Auckland, NZ, USA), respectively. Lentiviral NF2 shRNAs were from Sigma. NF2 shRNA1: 5′-CCGGCGGGCTTTGTTTCCTTCTTTACTCGAGTAAAGAAGGAAACAAAGCCCGTTTTTG-3′ NF2 shRNA2: 5′-CCGGGCTTCGTGTTAATAAGCTGATCTCGAGATCAGCTTATTAACACGAAGCTTTTTG-3′.
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