Bis tris plus bolt gels

Manufactured by Thermo Fisher Scientific

Sourced in United States

Bis-Tris Plus Bolt gels are precast polyacrylamide gels designed for protein electrophoresis. They provide a stable and consistent matrix for the separation of proteins based on their molecular weight.

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Lab products found in correlation

Perk t202 y204, by Cell Signaling Technology (2 mentions) P fak y397, by Cell Signaling Technology (2 mentions) Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions) Iblot dryblot system, by Thermo Fisher Scientific (2 mentions) P p38 t180 y182, by Cell Signaling Technology (2 mentions) P src y416, by Cell Signaling Technology (2 mentions) Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions) P akt, by Cell Signaling Technology (2 mentions) Ab10096, by Abcam (1 mentions) Nupage transfer buffer, by Thermo Fisher Scientific (1 mentions) Image studio, by LI COR (1 mentions) Odyssey system, by LI COR (1 mentions) Protein assay, by Bio-Rad (1 mentions) Ab9485, by Abcam (1 mentions) Bcl 6, by BD (1 mentions) Eomes, by Abcam (1 mentions) Mouse anti rabbit hrp, by Santa Cruz Biotechnology (1 mentions) Goat anti mouse hrp, by Jackson ImmunoResearch (1 mentions) β actin, by GenScript (1 mentions) Nupage, by Thermo Fisher Scientific (1 mentions)

6 protocols using bis tris plus bolt gels

Western Blot Analysis of Signaling Pathways

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Harvested cell culture pellets were resuspended in protein lysis buffer (25mM HEPES pH 7,5; 0,3M NaCl; 1,5mM MgCl2; 2mM EDTA; 2mM EGTA; 1mM DTT; 1% Triton X-100; 10% Glycerol; phosphatase/protease inhibitor cocktail), incubated on ice (15min) and centrifuged (15min) at 4°C/13000 rpm. Tumour samples were additionally homogenized with a PreCellys in protein lysis buffer, prior to incubation on ice. Equal amounts of protein (Bradford quantification) were run on 4-12% Bis-Tris Plus Bolt gels (ThermoFisherScientific) and transferred to a nitrocellulose membrane with an iBlot dryblot system (ThermoFisherScientific). Membrane blocking (5% BSA/TBS-0,2%Tween) is followed by incubation with the appropriate primary antibodies and HRP-conjugated secondary antibody (Cell Signaling). Signals were detected by enhanced chemiluminescence (ThermoFisherScientific) on Amersham hyperfilm. Antibodies were from Cell Signaling Technology (P-FAK Y397, #8556; FAK, #13009; P-SRC Y416, #6943; SRC, #2123; P-AKT S473, #4060; AKT, #4691; P-ERK T202/Y204, #9106 (cell lines); P-ERK T202/Y204 #4370 (tumour lysates); ERK, #9102; BRAF, #9433; P-p38 T180/Y182, #4511; p38, #8690; PTEN, #9559)

Marín-Béjar O., Rogiers A., Dewaele M., Femel J., Karras P., Poźniak J., Bervoets G., Raemdonck N.V., Pedri D., Swings T., Demeulemeester J., Pelt J.v., Bosisio F.M., Oord J.J.v.d., Bempt I.V., Lambrechts D., Voet T., Bechter O., Rizos H., Levesque M.P., Leucci E., Lund A.W., Rambow F., & Marine J. (2020). A neural crest stem cell-like state drives nongenetic resistance to targeted therapy in melanoma.

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Western Blot Analysis of Signaling Pathways

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Harvested cell culture pellets were resuspended in protein lysis buffer (25mM HEPES pH 7,5; 0,3M NaCl; 1,5mM MgCl2; 2mM EDTA; 2mM EGTA; 1mM DTT; 1% Triton X-100; 10% Glycerol; phosphatase/protease inhibitor cocktail), incubated on ice (15min) and centrifuged (15min) at 4 C/13000 rpm. Tumour samples were additionally homogenized with a PreCellys in protein lysis buffer, prior to incubation on ice. Equal amounts of protein (Bradford quantification) were run on 4-12% Bis-Tris Plus Bolt gels (ThermoFisher-Scientific) and transferred to a nitrocellulose membrane with an iBlot dryblot system (ThermoFisherScientific). Membrane blocking (5% BSA/TBS-0,2%Tween) is followed by incubation with the appropriate primary antibodies and HRP-conjugated secondary antibody (Cell Signaling). Signals were detected by enhanced chemiluminescence (ThermoFisherScientific) on Amersham hyperfilm. Antibodies were from Cell Signaling Technology (P-FAK Y397, #8556; FAK, #13009; P-SRC Y416, #6943; SRC, #2123; P-AKT S473, #4060; AKT, #4691; P-ERK T202/Y204, #9106 (cell lines); P-ERK T202/Y204 #4370 (tumour lysates); ERK, #9102; BRAF, #9433; P-p38 T180/Y182, #4511; p38, #8690; PTEN, #9559)

Marin-Bejar O., Rogiers A., Dewaele M., Femel J., Karras P., Pozniak J., Bervoets G., Van Raemdonck N., Pedri D., Swings T., Demeulemeester J., Borght S.V., Lehnert S., Bosisio F., van den Oord J.J., Bempt I.V., Lambrechts D., Voet T., Bechter O., Rizos H., Levesque M.P., Leucci E., Lund A.W., Rambow F, & Marine J.C. (2021). Evolutionary predictability of genetic versus nongenetic resistance to anticancer drugs in melanoma. Cancer cell, 39(8).

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Western Blot Analysis of α7nAChR

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The protein content of lysed cells was quantified using the Bio-Rad Protein Assay which is based on the Bradford Method (Bio-Rad, USA). Protein was reduced and prepared for western analysis using the Bolt LDS sample buffer and Reducing agent, before 10 µg protein was loaded into each well of a 10% Bis–Tris Plus Bolt Gels (Thermo Fisher Scientific, USA). Gels were run for 60 min at 150 v using NP MES SDS running buffer (Thermo Fisher Scientific, USA) and transferred to Nitrocellulose membrane at 10 v for 60 min using NuPAGE transfer buffer (Thermo Fisher Scientific, USA). Membranes were blocked with 5% milk in TBST before overnight incubation with the primary antibodies at 4 °C. The anti-ɑ7nAChR was used at 1/300 (ab10096, Abcam), while anti-GAPDH was used at 1/10000 (ab9485, Abcam). Proteins were then detected using IRDye 800CW-labeled antibodies and visualized on the Odyssey system (LI-COR Biosciences, USA). ɑ7nAChR was assessed relative to the GAPDH protein loading control using Image Studio (LI-COR Biosciences, USA) which included blot background correction.

Reid V.J., McLoughlin W.K., Pandya K., Stott H., Iškauskienė M., Šačkus A., Marti J.A., Kurian D., Wishart T.M., Lucatelli C., Peters D., Gray G.A., Baker A.H., Newby D.E., Hadoke P.W., Tavares A.A, & MacAskill M.G. (2024). Assessment of the alpha 7 nicotinic acetylcholine receptor as an imaging marker of cardiac repair-associated processes using NS14490. EJNMMI Research, 14, 7.

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Immunoblot Analysis of Cell Signaling

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Immunoblots were performed as described previously^{38 (link),77 (link)}. Briefly, cells were harvested and pellets were lysed and prepared via boiling for 15 min in 1X loading dye (50 mM Tris [pH 6.8], 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol). Lysates were separated by SDS-PAGE on 10% Bis-Tris Plus Bolt gels (ThermoFisher) and transferred onto 0.45 µm nitrocellulose membrane. Membranes were blocked with 2% nonfat dry milk in 1X TBST (10 mM Tris [pH 8], 150 mM NaCl, 0.05% Tween-20), and detection of indicated proteins was carried out using the following antibodies: Aiolos (39293, Active Motif, 1:20,000), Bcl-6 (clone K112, BD Biosciences, 1:500), pSTAT5_(Y694/9) (clone 47 BD Biosciences, 1:5000), STAT5 (clone (D206Y, Cell Signaling, 1:5000), β-Actin (GenScript, 1:15,000), Eomes (Abcam, 1:5,000) goat anti-mouse:HRP (Jackson Immunoresearch, 1:5,000-1:30,000), mouse anti-rabbit:HRP (Santa Cruz, 1:5000-1:20,000).

Read K.A., Jones D.M., Pokhrel S., Hales E.D., Varkey A., Tuazon J.A., Eisele C.D., Abdouni O., Saadey A., Leonard M.R., Warren R.T., Powell M.D., Boss J.M., Hemann E.A., Yount J.S., Xin G., Ghoneim H.E., Lio C.W., Freud A.G., Collins P.L, & Oestreich K.J. (2023). Aiolos represses CD4+ T cell cytotoxic programming via reciprocal regulation of TFH transcription factors and IL-2 sensitivity. Nature Communications, 14, 1652.

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Comprehensive Protein Extraction and Analysis

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Cells were lysed in RIPA buffer containing 150mM NaCl, 50mM Tris pH 8.0, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS supplemented with protease inhibitors (Complete, Roche, Switzerland) and phosphatase inhibitor cocktails II and III (Sigma-Aldrich, U.S.A.). 10X reducing agent and 4X LDS sample buffer (NuPAGE™, Thermo-Fisher Scientific, U.S.A.) were added. Subsequently, the samples were boiled for 5 minutes and quickly centrifuged at 21,000 rcf. Proteins were quantified using the BCA Protein Assay Kit (Pierce, Thermo-Fisher Scientific, U.S.A.). Equal amounts of protein were subjected to SDS gel electrophoresis using 4–12% Bis-Tris Plus Bolt gels (Thermo-Fisher Scientific, U.S.A.) and 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, followed by Western blotting. Western blotting was performed using antibodies from Cell Signaling (MA, U.S.A.) against total JAK3 (3775S), phospho-JAK3 (Tyr980/981, 5031S), STAT5 (9363S), phospho-STAT5 (Tyr694, 9351S), p44/42 MAPK (Erk1/2, 137F5, 4695S), P-p44/p42 MAPK (T202/Y204, 4370S), AKT (pan, C67E7, 4691L), phospho-AKT (S473, D9E, 4060L), GAPDH and antibody from Santa Cruz Biotechnology Inc. (U.S.A.) against HSP90 α/β (f-8, SC-13119). For the western blotting results representative examples of at least two independent experiments are reported.

Mittempergher L., Piskorz A.M., Bosma A.J., Michaut M., Wisman G.B., Kluin R.J., Nieuwland M., Brugman W., van der Ven K.J., Marass F., Morris J., Rosenfeld N., Jimenez-Linan M., de Jong S., van der Zee A.G., Brenton J.D, & Bernards R. (2020). Kinome capture sequencing of high-grade serous ovarian carcinoma reveals novel mutations in the JAK3 gene. PLoS ONE, 15(7), e0235766.

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CTIP2 Protein Detection in Aortic Tissue

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Frozen aortic tissues were homogenised using TissueLyser LT (QIAGEN) and
protein lysates were extracted in NE-PER nuclear and cytoplasmic extraction
buffers (ThermoFisher Scientific) containing protease inhibitors (Calbiochem).
Full-length CTIP2 expressed in a pcDNA3.1 vector and transfected into HEK293T
cells was used as a positive control for the antibody and empty vector
transfected into HEK293T cells was used as a negative control. Cells were lysed
using RIPA buffer and protein concentrations were determined with the PierceTM
BCA protein assay (ThermoFisher Scientific). Protein lysates (10ug) were
incubated a Laurel DuodecylSulfate (LDS) sample loading buffer and Bolt®
sample reducing agent (ThermoFisher Scientific) before loading and separation by
SDS-gel electrophoresis in 4-12% gradient Bis-Tris Plus Bolt® gels
(ThermoFisher Scientific) at 200V for 30 min. They were transferred to 0.22
μM nitrocellulose membrane (ThermoFisher Scientific) using the iBlot2 dry
blotting system (ThermoFisher Scientific) and blocked with 5% (wt/vol) milk in
TBS buffer then incubated with primary antibodies in TBS-Tween (0.1% Tween 20)
for 16 h at 4°C. Secondary antibodies were incubated in TBS-Tween for 1 h
at RT in the dark. Membranes were washed in TBS-Tween 3X for 15 min between
primary and secondary antibody incubations and visualized using the LI-COR
Odyssey system.

Maskari R.A., Hardege I., Cleary S., Figg N., Li Y., Siew K., Khir A., Yu Y., Liu P., Wilkinson I., O’Shaughnessy K, & Y.a.s.m.i.n. (2018). Functional characterization of common BCL11B gene desert variants suggests a lymphocyte-mediated association of BCL11B with aortic stiffness. European Journal of Human Genetics, 26(11), 1648-1657.

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