A two-hybrid assay was carried out as previously described (Gyuris et al. 1993 (link)). Atg21 was expressed as an activation domain (AD) fusion protein using plasmid pJG4-5-ATG21 (see Table S2). WT and mutant Rho5 proteins were expressed as DNA-binding domain (DBD) fusions using plasmids pEG202-RHO5 , pEG202-rho5 G12V, and pEG202-rho5 K16N, all of which also carry the C328S substitution, as previously described (Singh et al. 2008 (link)). The yeast strain EGY48 carrying the LEU2 reporter gene was transformed with pJG4-5-ATG21 and each of pEG202-RHO5 plasmids (or empty vector controls), and several independent transformants from each transformation were plated on SC-Gal plates lacking Leu (and Trp and His to maintain the plasmids) to test the Rho5 -Atg21 interaction. To compare the levels of WT or mutant Rho5 proteins fused to LexA, yeast extracts were prepared from the strain (EGY48) carrying each pEG plasmid and then were subjected to immunoblotting with rabbit polyclonal anti-LexA antibodies (EMD Millipore) and Alexa Fluor 680 goat anti-rabbit IgG (LI-COR Biosciences).
Alexa fluor 680 goat anti rabbit igg
Manufactured by LI COR
Sourced in United States
Alexa Fluor 680 goat anti-rabbit IgG is a secondary antibody conjugate. It is designed to detect and bind to rabbit primary antibodies, allowing for fluorescent labeling and detection.
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Lab products found in correlation
Odyssey clx imager, by LI COR (1 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (1 mentions)
Hmgb1 primary antibody, by Abcam (1 mentions)
Image studio 5, by LI COR (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
3 protocols using alexa fluor 680 goat anti rabbit igg
1
Two-Hybrid Assay for Atg21-Rho5 Interaction
2
HMGB1 Protein Detection in BALF
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Equal volumes of BALF were subjected to electrophoresis on NuPAGE 4-12% Bis-Tris gradient gel (Life Technologies, Carlsbad, CA), and transferred to PVDF membrane by using iBlot™ gel transfer device (Life Technologies, Carlsbad, CA). Then, the membrane was incubated with rabbit polyclonal HMGB1 primary antibody (ab18256; Abcam, Cambridge, MA). Alexa Fluor 680 goat anti-rabbit IgG (926-68071; LI-COR Biosciences, Lincoln, NE) was used as the secondary antibody. Protein bands were visualized using Odyssey CLx, Imager (LI-COR Biosciences, Lincoln, NE).
3
Protein Expression Analysis in Osteoclasts
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Total cellular proteins were extracted with Radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology, Shanghai, China) containing 10 mg/ml aprotinin, 10 mg/ml leupeptin and 100 mM PMSF. The bicinchoninic acid assay was used for protein quantification. The aliquots (containing 40 µg protein) were separated by 8% SDS-PAGE, transferred to polyvinyli-dene difluoride membrane. The membranes were incubated at 4°C overnight with antibodies against TRAP (cat. no. ab191406; Abcam; 1:1,000), NFATc-1 (cat. no. 8032; CST; 1:1,000), Cathepsin K (cat. no. ab187647; Abcam; 1:1,000), RANKL (cat. no. ab97864; Abcam; 1:1,000), JNK (cat. no. 9252; CST; 1:1,000) and p-JNK (cat. no. 4668; CST; 1:1,000). Anti-β-actin (cat. no. 4970; CST; 1:1,000) was used as a loading control. After washing, the membranes were incubated with IRDye® 800CW goat anti-Rabbit IgG (cat. no. 926-32211; LI-COR Biosciences, Lincoln, NE, USA; 1:10,000) or Alexa Fluor® 680 goat anti-rabbit IgG (cat. no. ab175773; Abcam; 1:10,000) for 1 h at room temperature. The target proteins were visual-ized and quantification was performed with the Odyssey Clx Infrared Imaging System (LI-COR Biosciences) and Image Studio 5.0 software (LI-COR Biosciences).
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