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Sds page 4 12 bis tris gels

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SDS PAGE 4–12% Bis-Tris gels are pre-cast polyacrylamide gels used for the separation of proteins based on their molecular weight. The gels have a gradient of 4-12% acrylamide, allowing for the separation of a wide range of protein sizes. They are designed for use in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques.

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Lab products found in correlation

Lsm 880 confocal microscope, by Zeiss (4 mentions) Ab10436, by Abcam (2 mentions) Iblot 2 dry blotting system, by Thermo Fisher Scientific (2 mentions) Quantity one software, by Bio-Rad (2 mentions) Proteinase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Nupage reagents and equipment, by Thermo Fisher Scientific (1 mentions) γh2ax ser139, by Santa Cruz Biotechnology (1 mentions) Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Ab150088, by Abcam (1 mentions) T9026, by Abcam (1 mentions) Mouse anti flag, by Merck Group (1 mentions) Total oxphos human wb antibody cocktail, by Abcam (1 mentions) Goat anti mouse igg hrp, by Promega (1 mentions) Goat anti rabbit igg hrp, by Promega (1 mentions) Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions) Goat anti rat hrp, by Santa Cruz Biotechnology (1 mentions) Goat anti mouse hrp, by Promega (1 mentions)

Close spelling variants of this product

NuPAGE 4–12% Bis-Tris Gels for SDS-PAGE (2 citations) Bolt SDS-PAGE 4%–12% Bis-Tris gels (6 citations) NuPAGE® Novex 4–12% (w/v) Bis-Tris SDS-PAGE gels (2 citations) 4-to-12% bis-Tris SDS-PAGE gels (4 citations) SDS–PAGE 4–12% Bis–Tris Protein Gels (2 citations) 4–12% Bis-Tris SDS-PAGE gels (89 citations) SDS-PAGE 4–12% gel (2 citations) Bis-Tris gels 4-12% (2 citations) Bis-Tris PAGE gel (14 citations) Bis-Tris SDS-PAGE (35 citations)

7 protocols using sds page 4 12 bis tris gels

1

Protein Extraction and Immunoblot Analysis

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Protein was extracted from cultured cells using RIPA-lysis buffer (50mM Tris-HCl pH8, 150mM NaCl, 1mM EDTA, 0.1% SDS, 0.5% Deoxycholate, 1% NP-40), supplemented with proteinase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA). The protein extracts were quantified using the DC protein assay (BioRad, Hercules, CA, USA). For immunoblot analysis, 70μg of protein was resolved by SDS PAGE 4–12% Bis-Tris gels (Life Technologies) and blotted to PVDF membrane (BioRad) using NuPAGE reagents and equipment (Life Technologies, Carlsbad, CA, USA). Blots were blocked in 5% BSA PBS-Tween buffer for 1h. Membranes were then incubated with primary antibodies recognizing androgen receptor-D6F11 (#5153, Cell Signaling, Danvers, MA, USA), γH2Ax-Ser139 (sc:101696, Santa Cruz Biotechnology, Dallas, TX, USA), or fibrillarin-H140 (sc:25397, Santa Cruz) overnight at 4°C. Blots were then washed and incubated with goat anti-rabbit IgG-HRP as secondary antibody for detection (Santa Cruz). Protein bands were then visualized using Super Signal WestFemto Reagent Substrate (Thermo Scientific) and developed on the AlphaInnotech FluorChem 8900 system (ProteinSimple, San Jose, CA, USA). Densitometry of each specific protein band was determined using AlphaInnotech FluorChem 8900 software and normalized to its corresponding fibrillarin band.
Palomera-Sanchez Z., Watson G.W., Wong C.P., Beaver L.M., Williams D.E., Dashwood R.H, & Ho E. (2017). The phytochemical 3,3′-Diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells. The Journal of nutritional biochemistry, 47, 113-119.
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2

Intracellular Localization of NSUN2 in 143B Cells

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The intracellular localization of NSUN2 by immunofluorescence in fixed human osteosarcoma 143B cells was performed as described previously (32 (link)). The following antibodies were used: rabbit anti-TOM20 (Santa Cruz Biotechnology, sc-1145, 1:200), rat anti-HA (Roche, 118667431001, 1:200), Alexa Fluor 594 anti-rabbit (1:1000, ab150088, Abcam), Alexa Fluor 488 anti rat (Abcam, ab150157, 1:1000), mouse anti-FLAG (Sigma, F1084, 1:500). Immunofluoresence images were captured using a Zeiss LSM880 confocal microscope and processed using ImageJ.
For western blot analysis, 10–30 μg of extracted proteins were loaded on SDS-PAGE 4–12% bis–tris gels (Life Technologies) and transferred onto a membrane using iBlot 2 Dry Blotting System (Thermo Fisher Scientific). The following antibodies were used: rabbit anti-NSUN2 (Proteintech, 20854-1-AP, 1:1000), mouse anti-Tubulin (Sigma Aldrich, T9026, 1:5000), mouse anti-TOM22 (Abcam, ab10436, 1:2000), mouse anti-GAPDH (Abcam, ab9484, 1:5000) mouse anti-FLAG (Sigma, F3165, 1:1000), mouse anti-mtSSB1 (a kind gift of Prof. D. Kang, 1:4000), total OXPHOS Mouse WB antibody cocktail (Abcam, ab110412, 1:2000) and mouse anti-HSC70 (Santa Cruz Biotechnology sc7298, 1:10 000).
Van Haute L., Lee S.Y., McCann B.J., Powell C.A., Bansal D., Vasiliauskaitė L., Garone C., Shin S., Kim J.S., Frye M., Gleeson J.G., Miska E.A., Rhee H.W, & Minczuk M. (2019). NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs. Nucleic Acids Research, 47(16), 8720-8733.
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3

Mitochondrial Ribosomal Protein Analysis

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20–30 µg of total extracted protein was diluted to an equal volume and a ⅓ volume of NuPAGE LDS 4 x sample buffer and loaded on SDS-PAGE 4-12% bis-tris gels (Life Technologies) and transferred onto a membrane using iBlot 2 Dry Blotting System (Thermo Fisher Scientific). The following antibodies were used: rabbit anti-MRPS17 (Proteintech 18,881-1-AP, 1:1000), rabbit anti-MRPS18b (Proteintech 16,139-1-AP, 1:1000), rabbit anti-MRPS35 (Proteintech 16,457-1-AP, 1:1000), Total OXPHOS Human WB antibody cocktail (Abcam, ab110411, 1:1000), goat anti-rabbit IgG HRP (Promega W4011, 1:2000), goat, anti-mouse IgG HRP (Promega W4021).
Powell C.A, & Minczuk M. (2020). TRMT2B is responsible for both tRNA and rRNA m5U-methylation in human mitochondria. RNA Biology, 17(4), 451-462.
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4

Immunofluorescence and Western Blot Analyses

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Localisation of proteins by immunofluorescence was carried out in fixed 143B cells as previously described (22 (link)). Images were captured using a Zeiss LSM 880 confocal microscope.
The following antibodies were used for immunofluorescence experiments in this work: mouse anti-TOM22 (Abcam, ab10436, 1:250), Alexafluor-594 anti-mouse (Molecular Probes, A11005, 1:1000), rabbit anti-TFAM (gifted by Prof. Rudolf Wiesner, 1:500), Alexafluor-405 anti-rabbit (Molecular Probes, A31553, 1:1000), rat anti-HA (Roche, 11867431001, 1:500), Alexafluor-488 anti-rat (Molecular Probes, A11006, 1:1000). Mounting medium used was either ProLong Gold Antifade Mountant (Molecular Probes), or ProLong Gold Antifade Mountant with DAPI (Molecular Probes).
For western blot analyses, ∼20 μg of extracted proteins were resolved on SDS-PAGE 4–12% bis-tris gels (Life Technologies). The following antibodies were used for western blotting in this work: mouse anti-FLAG (Sigma, F1804, 1:2000), rabbit anti-FLAG (Sigma, F7425, 1:2000), rat anti-HA (Roche, 11867431001, 1:1000), rabbit anti-Histone H4 (Abcam, ab10158, 1:5000), rabbit anti-SSB1 (kindly gifted by Prof D. Kang, 1:4000), mouse anti-TOM22 (Abcam, ab10436, 1:5000), mouse anti-GAPDH (Abcam, ab9484, 1:10 000), goat anti-rabbit HRP (Promega, W401B, 1:2000), goat anti-mouse HRP (Promega, W402B, 1:2000), goat anti-rat HRP (Santa Cruz, SC2065, 1:1000).
Gammage P.A., Gaude E., Van Haute L., Rebelo-Guiomar P., Jackson C.B., Rorbach J., Pekalski M.L., Robinson A.J., Charpentier M., Concordet J.P., Frezza C, & Minczuk M. (2016). Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs. Nucleic Acids Research, 44(16), 7804-7816.
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5

Protein Fractionation and Western Blot

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Nuclear and cytoplasmic protein fractions were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific). Protein lysates were denatured for 5 min with NuPAGE sample-reducing agent (LifeTech) and loading buffer heated to 75°C. Samples were subject ed to electrophoresis on SDS-PAGE 4–12% Bis-Tris gels (LifeTech). Proteins were electroblotted onto polyvinylidene fluoride membranes by using a Gel Transfer Device (Invitrogen). Membranes were dried, activated with methanol, blocked for 1 h at room temperature with Odyssey Blocking buffer diluted 1:2 in TBS, and incubated with the indicated antibodies at 4°C overnight. Odyssey secondary IR antibodies (diluted 1:10000) were used for detecting proteins with the Odyssey Infrared Imaging System.
Kullmann J.A., Trivedi N., Howell D., Laumonnerie C., Nguyen V., Banerjee S.S., Stabley D.R., Shirinifard A., Rowitch D.H, & Solecki D.J. (2020). Oxygen-tension and the VHL-Hif1α pathway determine onset of neuronal polarization and cerebellar germinal zone exit. Neuron, 106(4), 607-623.e5.
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6

ProMMP-7 Binding Affinity Assay

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ProMMP-7 (wt, E195A-inactivated, or with C-terminal KRSNSRKK deletion) was incubated with bovine HS (Sigma), low molecular weight heparin from porcine intestinal mucosa (Sigma), or the heparin oligosaccharides. The reaction mixtures were incubated for 2 or 3 h at 37°C, stopped using LDS sample buffer (Novex), separated on Bis-tris 4–12% SDS-PAGE gels (Novex), and stained with Coomassie Blue. The protein bands were scanned and quantified by Quantity One software (Biorad).
Fulcher Y.G., Gari R.R., Frey N.C., Zhang F., Linhardt R.J., King G.M, & Van Doren S.R. (2014). Heparinoids activate a protease, secreted by mucosa and tumors, via tethering supplemented by allostery. ACS chemical biology, 9(4), 957-966.
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7

Heparin Oligosaccharides Inhibit MMP-7 Activity

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ProMMP-7 were incubated with increasing concentrations of heparin oligosaccharides dp16 per (Fulcher et al., 2014 (link)). The reaction mixtures were incubated for 2 h at 37 °C, stopped using SDS-containing gel-loading buffer (Novex), separated on Bistris 4–12% SDS-PAGE gels (Novex), and stained with Coomassie Blue. The protein bands were quantified using Quantity One software (Biorad).
Fulcher Y.G., Prior S.H., Masuko S., Li L., Pu D., Zhang F., Linhardt R.J, & Van Doren S.R. (2017). Glycan Activation of a Sheddase: Electrostatic Recognition between Heparin and proMMP-7. Structure (London, England : 1993), 25(7), 1100-1110.e5.
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