Tgx gradient gel

Manufactured by Bio-Rad

The 4–20% TGX gradient gel is a polyacrylamide gel used for the separation and resolution of proteins in electrophoresis. It provides a continuous gradient of 4% to 20% polyacrylamide concentration, allowing for the effective separation of a wide range of protein molecular weights.

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Lab products found in correlation

Pvdf membrane, by Merck Group (5 mentions) Bca assay, by Thermo Fisher Scientific (4 mentions) Trans blot turbo pvdf nitrocellulose membrane, by Bio-Rad (4 mentions) Supersignal west dura substrate, by Thermo Fisher Scientific (4 mentions) Complete ultra mini, by Roche (3 mentions) β actin, by Cell Signaling Technology (3 mentions) Clarity western ecl substrate, by Bio-Rad (3 mentions) Trans blot turbo transfer system, by Bio-Rad (3 mentions) Chemidoc mp system, by Bio-Rad (3 mentions) Imagequant las 4000, by GE Healthcare (3 mentions) Las 4000, by Cytiva (3 mentions) Nb400 105, by BD (2 mentions) Theodyssey imaging system, by LI COR (2 mentions) Protease and phosphatase inhibitor, by Roche (2 mentions) Ripa buffer, by Cell Signaling Technology (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Merck Group (2 mentions) Nitrocellulose membrane, by Cytiva (2 mentions) Full range rainbow prestained protein standards, by GE Healthcare (2 mentions) Western lightning enhanced chemiluminescence, by PerkinElmer (2 mentions)

Spelling variants of this product

4–20% gradient gels (350 citations) TGX gels (272 citations) 4–20% gradient TGX gels (22 citations) Mini Protean TGX 4-15% gradient gels (3 citations) Mini protean TGX precast 4-20% gradient gels (3 citations) Mini-PROTEAN TGX™ 4 to 15% gradient gels (2 citations) Gradient Mini-PROTEAN TGX precast protein gels (2 citations) Mini-PROTEAN TGX 5–20% gradient gels (2 citations) Mini-PROTEIN TGX 4–20% gradient gels (2 citations) 4–15% gradient TGX precast gels (2 citations)

44 protocols using tgx gradient gel

Western Blot Analysis of ABCA1 and HSP90

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Protein was extracted in RIPA buffer (Cell Signaling) with protease and
phosphatase inhibitors (Roche) and subsequently normalized by Pierce BCA Protein
Assay Kit (Thermo Fisher Scientific). Samples (30 μg/well) were
electrophoresed on 4–20% TGX-gradient gels (Bio-Rad Laboratories) and
transferred to nitrocellulose membranes at 125 V for 2 h. Membranes were
incubated overnight with specified antibodies directed against ABCA1 (Novus
Biologicals, NB400–105) and HSP90 (BD Biosciences, #610419). Proteins
were visualized using appropriate secondary antibodies and scanned using the
Odyssey Imaging System (Li-Cor Biosciences). Quantification was performed using
Image Studio software (Li-Cor Biosciences).

Hennessy E.J., van Solingen C., Scacalossi K.R., Ouimet M., Afonso M.S., Prins J., Koelwyn G.J., Sharma M., Ramkhelawon B., Carpenter S., Busch A., Chernogubova E., Matic L.P., Hedin U., Maegdefessel L., Caffrey B.E., Hussein M.A., Ricci E.P., Temel R.E., Garabedian M.J., Berger J.S., Vickers K.C., Kanke M., Sethupathy P., Teupser D., Holdt L.M, & Moore K.J. (2018). The long noncoding RNA CHROME regulates cholesterol homeostasis in primate. Nature metabolism, 1(1), 98-110.

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Quantitative Muscle Protein Analysis

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Total protein concentrations for whole muscle lysates were determined by bicinchoninic acid assay, and equal amounts of protein for each sample were separated via SDS-PAGE, and transferred to polyvinyl difluoride membranes. Aliquots of heated (95–100 °C) and non-heated single fiber lysates were separated by SDS-PAGE using 4–20% TGX gradient gels (#456–1096: Bio-Rad, Hercules, CA) or 10% gels, and then transferred to polyvinyl difluoride membranes. After electrotransfer, gels were stained in SimplyBlue™ SafeStain for 1 h at room temperature and then destained with deionized water for another 2 h. The SimplyBlue-stained MHC bands on the gels were quantified by densitometry (AlphaView; ProteinSimple, San Leandro, CA) and served as the loading controls for the subsequently immunoblotted proteins^{42 (link),43 (link)}. Membranes were incubated with appropriate concentrations of primary and secondary antibodies, and subjected to enhanced chemiluminescence (Luminata Forte Western HRP Substrate; #WBLUF0100; Millipore) to quantify protein bands by densitometry (FluoroChem E Imager, AlphaView software; ProteinSimple, San Leandro, CA). Individual values were normalized to the mean value of all samples on the membrane and divided by the corresponding MHC loading control value.

Pataky M.W., Wang H., Yu C.S., Arias E.B., Ploutz-Snyder R.J., Zheng X, & Cartee G.D. (2017). High-Fat Diet-Induced Insulin Resistance in Single Skeletal Muscle Fibers is Fiber Type Selective. Scientific Reports, 7, 13642.

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Protein Extraction and Immunoblot Analysis

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Cells were lysed in RIPA buffer (Sigma) supplemented with 1% protease inhibitor ‘cocktail’ (Pierce). Proteins were then separated by electrophoresis through 4%–20% TGX gradient gels (BioRad) and were transferred to polyvinylidene fluoride membranes (Immobilion; Millipore). For the detection of human proteins by immunoblot analysis, we used primary antibodies listed in online supplemental table S5 and western enhanced chemiluminescence (BioRad). Blots were developed in ChemiDoc MP imaging system (BioRad).

Kumar A., Taghi Khani A., Duault C., Aramburo S., Sanchez Ortiz A., Lee S.J., Chan A., McDonald T., Huang M., Lacayo N.J., Sakamoto K.M., Yu J., Hurtz C., Carroll M., Tasian S.K., Ghoda L., Marcucci G., Gu Z., Rosen S.T., Armenian S., Izraeli S., Chen C.W., Caligiuri M.A., Forman S.J., Maecker H.T, & Swaminathan S. (2023). Intrinsic suppression of type I interferon production underlies the therapeutic efficacy of IL-15-producing natural killer cells in B-cell acute lymphoblastic leukemia. Journal for Immunotherapy of Cancer, 11(5), e006649.

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Western Blot Protein Analysis

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Cells were lysed in RIPA buffer (Sigma) supplemented with 1% protease inhibitor ‘cocktail’ (Pierce). Proteins were separated by electrophoresis through 4–20% TGX gradient gels (BioRad) and transferred to polyvinylidene fluoride membranes (Immobilon; Millipore). Mouse and human proteins were detected by immunoblotting using antibodies (Supplementary Table 2b) and western enhanced chemiluminescence (BioRad).

Taghi Khani A., Kumar A., Sanchez Ortiz A., Radecki K.C., Aramburo S., Lee S.J., Hu Z., Damirchi B., Lorenson M.Y., Wu X., Gu Z., Stohl W., Sanz I., Meffre E., Müschen M., Forman S.J., Koff J.L., Walker A.M, & Swaminathan S. (2023). Isoform-specific knockdown of long and intermediate prolactin receptors interferes with evolution of B-cell neoplasms. Communications Biology, 6, 295.

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Western Blot Analysis of ABCA1 and HSP90

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Western Blot Analysis of Exosomal Markers

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Whole cell lysates were prepared by lysing HFKs on ice for 40 min in 1X Tris-EDTA buffer with 1% NP40 Alternative (Millipore), containing protease and phosphatase inhibitors (Roche). Insoluble protein and membranes were pelleted by centrifugation at 20,000 xG for 30 min at 4 °C. Total protein in lysates was measured by BCA assay (Thermo-Fisher), separated through 4–20% TGX gradient gels (Bio-Rad), electro-transferred onto PVDF membranes (Millipore), and blocked in BSA blocking buffer (Li-Cor) at room temperature for one hour. Membranes were probed overnight for Alix (Bio-Rad), TSG101 (Abcam), LC3b (Thermo-Fisher), IL36γ (R&D), CD63 (Bioss) or β-actin (Sigma). Membranes were then washed, probed with near-infrared dye-conjugated secondary antibodies donkey anti-mouse CW800, donkey anti-rabbit CW800, or donkey anti-goat 680RD (Li-Cor). Blots were scanned and quantified using Azure Biosystems Sapphire Biomolecular imager.

Papayannakos C.J., DeVoti J.A., Israr M., Alsudani H., Bonagura V, & Steinberg B.M. (2021). Extracellular vesicles produced by primary human keratinocytes in response to TLR agonists induce stimulus-specific responses in antigen-presenting cells.. Cellular signalling, 83, 109994.

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Immunoprecipitation and Western Blot Analysis

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Cells harvested from the transfected cells used in the tethered function assays were harvested in 1× PBS, pelleted, and resuspended in radioactive immunoprecipitation assay buffer (RIPA buffer, 50 mM Tris (pH 8.0), 150 mM NaCl, 1% (v/v) IGEPAL CA-630, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulphate) for lysis. Lysates were homogenized using QIAShredder Columns (Qiagen) and sample concentrations were determined using the BioRad DC Lowry assay. Each sample (8 μg total protein) was resolved on 4–20% TGX gradient gels (BioRad) and then transferred to Millipore EMD Immobilon membrane. Transfected NOCT proteins were detected using anti-V5 antibody (Invitrogen, R960–25) and HT was detected using anti-HT antibody (Promega, G9211). Blots were probed using HRP-conjugated Secondary Antibody (Sigma, A1047) and Pierce ECL Western Blotting substrate before exposure to autoradiography film. As a loading control, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected on the same blots using anti-GAPDH antibody (Ambion, AM4300).

Abshire E.T., Chasseur J., Bohn J.A., Del Rizzo P.A., Freddolino P.L., Goldstrohm A.C, & Trievel R.C. (2018). The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells. Nucleic Acids Research, 46(12), 6257-6270.

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Protein Extraction and Western Blot Analysis

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Total proteins were prepared by resuspending cells in RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.1% sodium dodecyl sulphate (SDS), 0.5% sodium deoxycholate, 1% NP-40, 1 mM EDTA, protease and phosphatase inhibitors [Roche]). Protein concentration was determined using a BCA assay (ThermoFisher Scientific). The cell lysate was denatured at 95°C for 5 min. The cell lysates were resolved on 4–15% TGX gradient gels (Bio-Rad Laboratories) and transferred to nitrocellulose membrane (Amersham). Membranes were blocked with 5% non-fat dried milk in TBS with 0.2% Tween for 1 hr at RT and then incubated with primary antibody overnight at 4°C. Primary antibodies used were against MF20 (MyHC) (DSHB, Mf20-s; RRID:AB_2147781), G9a (Abcam, ab185050; RRID:AB_2792982), Ezh2 (Cell Signaling, #3147; RRID:AB_10694383), Wnt7b (Abcam, ab94915; RRID:AB_10675749), Cyclin D3 (Santa Cruz biotechnology, sc-182; RRID:AB_2259653), and GAPDH (Sigma, G9545; RRID:AB_796208). After washing in TBS with 0.2% Tween, membranes were incubated in HRP-conjugated specific secondary antibody (goat anti-rabbit-anti-mouse [IgG-HRP Santa Cruz Biotechnologies]) for 1 hr at RT. After washing in TBS with 0.2% Tween, blots were developed with Western lightning enhanced chemiluminescence (ThermoFisher Scientific), the signal detection was performed with the use of ChemiDoc (Bio-Rad).

Cipriano A., Macino M., Buonaiuto G., Santini T., Biferali B., Peruzzi G., Colantoni A., Mozzetta C, & Ballarino M. (2021). Epigenetic regulation of Wnt7b expression by the cis-acting long noncoding RNA Lnc-Rewind in muscle stem cells. eLife, 10, e54782.

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Immunoblotting Proteins with Diverse Antibodies

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Proteins were separated by SDS-PAGE on 4–15% TGX gradient gels (Bio-Rad) and transferred onto PVDF membranes. The membranes were blocked in PBS containing 5% non-fat milk and 0.1% Tween 20 for 30 min at room temperature. Blots were probed with the following antibodies: rabbit anti-eIF4E2/4EHP (Proteintech, 12227-1-AP), rabbit anti-DDX6 (Proteintech, 14632-1-AP), rabbit anti-CNOT9 (Proteintech, 22503-1-AP), mouse anti-Flag M2 (Sigma-Aldrich, F1804), rabbit anti-GIGYF2 (Proteintech, 24790-1-AP), mouse anti-GAPDH (Proteintech, 60004-1-Ig), mouse anti-V5 tag (Thermo Fisher Scientific, R96025), rabbit anti-ZNF598 (Thermo Fischer Scientific, 703,601) and mouse anti-6xHis Tag (Thermo Fischer Scientific, MA1-21315-HRP).

Zou L., Moch C., Graille M, & Chapat C. (2022). The SARS-CoV-2 protein NSP2 impairs the silencing capacity of the human 4EHP-GIGYF2 complex. iScience, 25(7), 104646.

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Western Blot Analysis of Autophagy and SMN Proteins

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Cells and tissues were lysed in 8M urea lysis buffer (150 mM NaCl, 50 mM Tris, 1% NP40, 8M Urea supplemented with EDTA-free SigmaFast protease inhibitor cocktail). 20ug total protein per lane was run on 4–15% TGX gradient gels (Biorad) and transferred to PVDF membranes. Rabbit polyclonal p62, and LC3 antibodies (Sigma) were used at 1:3000 and 1:10,000 respectively. Rabbit polyclonal SMN antibody (SantaCruz) was used at 1:1000. Mouse monoclonal FLAG (M2) antibody and mouse monoclonal tubulin and actin antibodies (Sigma) were used at 1:10,000 or 1:5000.

Custer S.K, & Androphy E.J. (2014). Autophagy dysregulation in cell culture and animals models of Spinal Muscular Atrophy. Molecular and cellular neurosciences, 0, 133-140.

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