Protein samples were prepared from cells using standard methods and were subjected to SDS-PAGE and transferred to a nitrocellulose membrane by wet western blot transfer (Bio-Rad) [12 (link)]. The membrane was blocked in TBST buffer (TBS + 1% Tween) containing 5% milk for 1 hour at room temperature. The blocked membranes were incubated overnight at 4°C with specific primary antibodies (Odyssey blocking buffer, 927–40000). The membranes were washed 3 times with TBST buffer, and then incubated with specific secondary antibodies provided by LI-COR for 2 hours at room temperature. After 3 washes with TBST buffer, the membranes were analyzed using the ODYSSEY Infrared Imaging system (LI-COR). Analysis of biotinylated cell surface proteins was performed using the Pierce Cell Surface Protein Isolation Kit (89881) from Thermo Scientific (Waltham, MA) [12 (link)]. Briefly, cells were labeled with Sulfo-NHS-SS-Biotin, a thiol-cleavable amine-reactive biotinylation reagent, and then lysed with mild detergent. Biotinylated surface proteins were isolated with Avidin Agarose, and eluted using SDS-PAGE sample buffer containing 50mM DTT. Samples were then analyzed for HER2 by immunoblot as above. All immunoblot experiments were performed at least 3 times and representative blots are shown in the figures.
Specific secondary antibodies
Manufactured by LI COR
Specific secondary antibodies are laboratory reagents used to detect and visualize target proteins in samples. They are designed to bind to the constant region of primary antibodies, allowing for the amplification and detection of the target protein signal. These secondary antibodies are highly specific and can be conjugated with various reporter molecules, such as fluorescent dyes or enzymes, to enable sensitive and accurate protein detection.
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Lab products found in correlation
3 protocols using specific secondary antibodies
1
Protein Isolation and Western Blot Analysis
2
Immunoblotting of Protein Samples
3
Immunoblotting of Protein Samples
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Protein samples were prepared from cells using standard methods, were subjected to SDS-PAGE and transferred to a nitrocellulose membrane by wet Wwestern blot transfer (Bio-Rad). The membrane was blocked in TBST buffer (TBS + 1% Tween) containing 5% milk for 1 hour at room temperature. The blocked membranes were incubated overnight at 4°C with specific primary antibodies (Odyssey blocking buffer, 927-40000). The membranes were washed 3 times with TBST buffer, and then incubated with specific secondary antibodies provided by LI-COR for 2 hours at room temperature. After 3 washes with TBST buffer, the membranes were analyzed using the ODYSSEY Infrared Imaging system (LI-COR). All immunoblot experiments were performed at least 3 times and representative blots are shown in the figures. Primary antibodies included those against: phospho-HER2 (thr1221/1222) (2243S), EGFR (4267S), AKT (4691S), phospho-AKT (4060S) from Cell Signaling (Danvers, MA); HER2 (sc-33684), phospho-EGFR (sc-12351), flotillin1 (sc-25506) from Santa Cruz (Dallas, Texas); PMCA2 (PA1-915) from Thermo Scientific (Waltham, MA).
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