Detection reagents for yeast display were mouse-anti-c-MYC (clone 9E10, 13–2500), goat anti-chicken-AF 488 (A-11039), goat anti-mouse-AF 488 (A-11001), and Streptavidin-AF 647 (S-32357) from Life Technologies; chicken-anti-c-MYC (ACMYC) from Gallus Immunotech Inc.; and anti-His-AF 647 (35370) from Qiagen. Western blot antibodies were anti-vinculin (13901S), anti-B-Raf (14814S), anti-pMEK1/2 (Ser217/221) (9121S), anti-MEK1/2 (9122S), anti-pERK p44/42 (Thr202/Tyr204) (9101S), anti-ERK (9102S), anti-pAKT (Ser473) (9271S), and anti-AKT (9272S) from Cell Signaling Technologies, and anti-HA (16B12) from BioLegend. Blots were detected with HRP-conjugated secondaries (406401 and 405306) from BioLegend. GppNHp and GDP were purchased from Abcam, Jena Bioscience, and Sigma-Aldrich.
Anti ha 16b12
Manufactured by BioLegend
Anti-HA (16B12) is a mouse monoclonal antibody that specifically recognizes the hemagglutinin (HA) epitope tag. The HA tag is a commonly used reporter tag for recombinant protein expression and detection.
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Lab products found in correlation
4 protocols using anti ha 16b12
1
Yeast Display Reagents and Detection
2
Protein Quantification and Visualization
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Cells were solubilized in a RIPA buffer supplemented with 1 mM PMSF, 5 μg/mL leupeptin, and 5 μg/mL pepstatin A. Equal amounts of proteins in cell lysate were analyzed by a Western blot using anti-HA (16B12, BioLegend, San Diego, CA, Cat# 901515), anti-HiBiT (Promega, Cat# N7200) antibodies or HRP-Neutravidin (HRP-NA, ThermoFisher, Cat #A2664) as previously (Okiyoneda et al., 2010 (link); Okiyoneda et al., 2018 (link)). Western blots were visualized using a SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher) or ImmunoStar Zeta (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and analyzed by FUSION Chemiluminescence Imaging System (Vilber Bio Imaging, France). The staining of Ponceau S (Sigma-Aldrich, St Louis, MO) was used as a loading control.
3
Alkaline Protein Extraction for Western Blot
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The sample preparation for Western blotting was performed using an alkaline protein extraction method41 (link). Anti-GFP (7.1 and 13.1, Roche), anti-HA (16B12, BioLegend) or anti-Cdc2 (Y100.4, Santa Cruz) antibodies were used for probing Rec8-GFP, HA-Rec8 and Cdc2, respectively.
4
Alkaline Protein Extraction for Western Blot
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