Polyacrylamide gel electrophoresis was performed on pre-cast NuPAGE™ 4–12% Bis-Tris Midi Protein Gels (Invitrogen) polyacrylamide gels. Protein samples analysed under reducing conditions were incubated with 250 mM DTT before loading onto the gel. Gels were electrophoresed in NuPAGE™ MES SDS Running Buffer (Invitrogen). Total protein in a gel was visualised by Coomassie staining (Quick Coomassie, Generon). Proteins were blotted on nitrocellulose membranes using the BioRad TransBlot Turbo Transfer System (BioRad). Different mouse and rat antibodies at a concentration of 1 µg/ml were bound to the membrane using the iBind Flex western system (Invitrogen), alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, A8438, 1:1000) or alkaline phosphatase-conjugated goat anti-rat IgG (Sigma, A8438, 1:1000) were used for detection. Western blots were developed using SIGMAFAST™ BCIP®/NBT (Sigma).
Nupage 4 12 bis tris midi protein gels
Manufactured by Thermo Fisher Scientific
The NuPAGE™ 4–12% Bis-Tris Midi Protein Gels are precast polyacrylamide gels designed for the separation of proteins using the NuPAGE® electrophoresis system. The gels have a gradient of 4-12% acrylamide, which allows for the separation of a wide range of protein molecular weights. The Bis-Tris buffer system provides a stable pH environment for protein separation.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (2 mentions)
Sigmafast bcip nbt, by Merck Group (2 mentions)
Alkaline phosphatase conjugated goat anti rat igg, by Merck Group (1 mentions)
Quick coomassie, by Generon (1 mentions)
Ibind flex western system, by Thermo Fisher Scientific (1 mentions)
Alkaline phosphatase conjugated goat anti mouse igg, by Merck Group (1 mentions)
Luminata crescendo chemiluminescence western blotting substrate, by Merck Group (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Amersham imager 600rgb, by GE Healthcare (1 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Goat anti mouse igg alkaline phosphatase antibody, by Merck Group (1 mentions)
Nupage reducing agent, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Immun blot low fluorescence pvdf membrane, by Bio-Rad (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey imager, by LI COR (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
5 protocols using nupage 4 12 bis tris midi protein gels
1
Protein Expression Analysis via SDS-PAGE and Western Blotting
2
Western Blot Analysis of PAICS Protein
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Protein samples were resolved on NuPAGE 4%-12% Bis-Tris Midi Protein Gels (Invitrogen, Carlsbad, CA) and transferred onto Immobilon-P PVDF membranes (EMD Millipore, Billerica, MA) as described previously [30 (link)]. Immunoreactivity was evaluated by probing the membranes with mouse primary antibody followed by incubation with secondary IgG horseradish peroxidase antibody (Table S1). Immunoreactivity against PAICS protein was evaluated by use of Luminata Crescendo chemiluminescence Western blotting substrate according to the manufacturer's protocol (EMD Millipore), and images were captured with an Amersham Imager 600RGB (GE Healthcare Life Sciences, Pittsburgh, PA).
3
SDS-PAGE and Western Blot Analysis of Vaccine Proteins
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Vaccine samples were prepared in Laemmli buffer (BioRad) and proteins separated by SDS-PAGE on pre-cast NuPAGE 4-12% Bis-Tris Midi Protein Gels (Invitrogen) using NuPAGE MES SDS Running Buffer (Invitrogen). Total protein was either visualized by silver staining (Pierce Silver Stain Kit, Thermo Scientific) or blotted on nitrocellulose membranes using the BioRad TransBlot Turbo Transfer System (BioRad). Fusion proteins were detected using mouse anti-HBsAg (BioRad, MCA4658) and goat anti-mouse IgG-Alkaline Phosphatase antibody (Sigma, A3562). Western blots were developed with SIGMAFAST BCIP/NBT (Sigma).
4
Western Blot Analysis of Cell Lysates
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Cell or mouse tumor samples were lysed using RIPA lysis buffer: 0.5% Sodium deoxycholate, 50 mM Tris-HCl pH 7.4, 1% Triton, 137 mM NaCl, 1% Glycerin, 0.5 mM EDTA pH 8.0, 0.1% SDS. Following protein concentrations determination using the BCA Protein Assay Kit (Pierce, Appleton, WI), cell lysates were mixed with 4x NuPAGE LDS Sample Buffer (Invitrogen) and 10x NuPAGE Reducing Agent (Invitrogen) and heated at 98°C for 10 min. Lysates were separated using NuPAGE 4–12% BisTris Midi Protein gels (Invitrogen) and NuPAGE MOPS SDS Running Buffer (Invitrogen). Proteins were subsequently transferred onto Immun-Blot Low Fluorescence PVDF membranes (Bio-Rad) using the Bio-Rad Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked using the Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE) on a rocking platform for 1 h. Primary antibodies (refer to key resources table ) were incubated at 4°C overnight. Following washing steps, membranes were incubated with secondary antibodies (refer to key resources table ) for 1 h at RT. Signals were detected using the Odyssey Imager (LI-COR Biosciences, Model 9120).
5
Western Blot Analysis of Cellular Signaling
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Cell lysates were prepared in RIPA buffer (Sigma Aldrich # R0278) supplemented with dithiothreitol (Life Technologies # 15508013), phenylmethane sulfonyl fluoride (Sigma Aldrich # P7626), protease inhibitor cocktail (Sigma Aldrich # P8340) and phosphatase inhibitor tablets (Roche # 4906845001). NuPAGE 4-12% Bis-Tris Midi protein gels (Invitrogen # WG1402A) were used for western blot. After electrophoresis, gel was transferred using Trans-Blot Turbo PVDF or nitrocellulose transfer pack (Bio-Rad # 1704158 and # 1704156) . Membranes were probed with specific primary antibodies at 4 °C overnight and then incubated with corresponding HRP-conjugated secondary antibodies. We use Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific # 32132X3) as developing reagent.
Primary antibodies used in this study are: FOXO3 (CST #2497S), CDKN1B (CST #3698S), p-AKT (CST #12694S), pS6 (CST #4857S), MCL (CST #5453S), PARP (CST #9532S), cleaved PARP (CST #5625S), Actin (C4) (SC-47778). Secondary antibodies are from Invitrogen (anti-mouse A16090, anti-rabbit 32260, anti-goat 81-1620).
Primary antibodies used in this study are: FOXO3 (CST #2497S), CDKN1B (CST #3698S), p-AKT (CST #12694S), pS6 (CST #4857S), MCL (CST #5453S), PARP (CST #9532S), cleaved PARP (CST #5625S), Actin (C4) (SC-47778). Secondary antibodies are from Invitrogen (anti-mouse A16090, anti-rabbit 32260, anti-goat 81-1620).
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