Cultured cells were lysed in RIPA buffer on ice for 10 min. After 10-min centrifugation at 4 °C, 20,000 × g, whole cell lysates were mixed with 4X LDS sample buffer (Invitrogen) and denatured at 95 °C for 5 min. Samples were loaded on a 4–12% Bis-Tris SDS-PAGE gel, run at 200 V for 45 min in MOPS buffer, and transferred onto a nitrocellulose membrane (Bio-Rad) in an XCell II Blot module (Invitrogen) (15 V, 45 min). After 1-h blocking with 5% nonfat dry milk in PBST, the membrane was incubated with primary antibodies (rabbit anti-UPF1, Cell Signaling Technology #12040; mouse anti-G3BP1, Millipore #05–1938; mouse anti-puromycin clone 12D10, Millipore #MABE343; rabbit anti-hnRNP H, Bethyl Laboratories #A300-511A; chicken anti-GFP, Aves Labs #1010; rabbit anti-phospho-UPF1, Millipore #07-1016; mouse anti-β-Actin, Cell Signaling Technology #4967; rabbit anti-G3BP2, Bethyl Laboratories #A302-040A; rabbit anti-BFP, Evrogen #AB233) diluted (1:2000) in 5% milk/PBST at 4 °C with slow shaking overnight. After incubation, membranes were rinsed three times with PBST, and incubated with IR680- or IR800-conjugated secondary antibodies (Li-Cor) diluted (1:10,000) in 5% milk/PBST at room temperature for 1 h. After three rinses with PBST, membranes were imaged using an Odyssey CLx system (Li-Cor).
Ir680 or ir800 conjugated secondary antibodies
Manufactured by LI COR
IR680- or IR800-conjugated secondary antibodies are fluorescently-labeled antibodies that can be used to detect and visualize target proteins in various applications, such as Western blotting, immunohistochemistry, and cell-based assays. These antibodies are designed to specifically bind to primary antibodies, allowing for the detection and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Lds sample buffer, by Thermo Fisher Scientific (3 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (2 mentions)
Odyssey clx system, by LI COR (2 mentions)
Mouse anti β actin, by Cell Signaling Technology (1 mentions)
Rabbit anti upf1, by Cell Signaling Technology (1 mentions)
Mouse anti g3bp1, by Merck Group (1 mentions)
Rabbit anti g3bp2, by Fortis Life Sciences (1 mentions)
Chicken anti gfp, by Aves Labs (1 mentions)
Rabbit anti c19orf66, by Thermo Fisher Scientific (1 mentions)
Vinculin, by Merck Group (1 mentions)
Pan cadherin, by Merck Group (1 mentions)
Flag tag (flag), by Rockland Immunochemicals (1 mentions)
Gfp 1020, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
3 protocols using ir680 or ir800 conjugated secondary antibodies
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of C19ORF66 in HEK293T Cells
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Transfected HEK293T cells were lysed in RIPA buffer on ice for 10 min. After 10 min centrifugation at 4 °C, 20,000 × g, whole cell lysates were mixed with 4X LDS sample buffer (Invitrogen) and denatured at 95 °C for 5 min. Samples were loaded on a 4–12% Bis-Tris SDS-PAGE gel, run at 200 V for 45 min in MOPS buffer, and transferred onto a nitrocellulose membrane (Bio-Rad) in an XCell II Blot module (Invitrogen) (15 V, 45 min). After 1-hour blocking with 5% nonfat dry milk in PBST, the membrane was incubated with primary antibodies (rabbit anti-C19ORF66, Invitrogen # PA5–59815; mouse anti-β-Actin) diluted (1:2000) in 5% milk/PBST at 4 °C with slow shaking overnight. After incubation, membranes were rinsed three times with PBST, and incubated with IR680- or IR800-conjugated secondary antibodies (Li-Cor) diluted (1:10,000) in 5% milk/PBST at room temperature for 1 hour. After three rinses with PBST, membranes were imaged using an Odyssey CLx system (Li-Cor).
3
Western Blot Protein Detection
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Cultured cells were lysed in RIPA buffer on ice for 10 min. After 10 min centrifugation at 4°C, 20,000 g, whole-cell lysates were mixed with 4X LDS sample buffer (Thermo Fisher) and denatured at 95°C for 5 min. Samples were loaded on a 4–12% Bis-Tris-SDS-PAGE gel, run at 120 V for 2 h 30 min in MOPS buffer, and transferred onto a nitrocellulose membrane (Bio-Rad) at 15V for 55 min. After 1 h blocking with 5% nonfat dry milk in TBS-T, the membrane was incubated with primary antibodies against protein of interest (NPR, Santa Cruz, sc-39008, 1:100; GFP, Aveslab, GFP-1020, 1:5,000; FLAG, Sigma, F1804, 1:1,000; FLAG, Rockland, 600-401-383, 1:1,000; GAPDH, Sigma, G9545, 1:2,000; Pan-Cadherin, Sigma, C1821, 1:1,000; Vinculin, Sigma, V9264, 1:5,000) diluted in 5% milk/TBS-T, placed on a shaker overnight at 4°C. After incubation, membranes were washed three times with TBS-T, followed by incubation with IR680- or IR800-conjugated secondary antibodies (Li-Cor, 1:5,000) diluted in 5% milk/TBS-T at room temperature for 1 h. Subsequently, membranes were washed with TBS-T and imaged using Odyssey XF system (Li-Cor, 2800).
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