Protein levels of GFP, dnRhoA, and β-Actin were assessed using Western Blot, following previously described protocols.27 (link) Briefly, 5 μg of protein were separated on pre-cast SDS-PAGE gels (Bio-Rad) and transferred onto Immobilon IP membranes (Millipore) before probing for GFP (1:2500, Abcam, #ab6556), RhoA (1:500, #MA1-134, Thermo Fisher Scientific), β-Actin (1:2500, #ab170325, Abcam), and GAPDH (1:50,000, #PA1-9046, Thermo Fisher Scientific). Primary antibodies were detected using IRDye 800CW goat anti-mouse (#925-332210), IRDye 680RD donkey anti-rabbit (#925-68073), or IRDye 800CW donkey anti-goat (#926-32214) secondary antibodies (1:15,000, LI-COR). Blots were visualized on an Odyssey Infrared Imaging System (LI-COR). Blots were stripped with NewBlot PVDF Stripping Buffer (LI-COR) and reprobed once to allow for detection of all four proteins on the same blot.
Ab170325
Manufactured by Abcam
Ab170325 is a lab equipment product manufactured by Abcam. It is a versatile tool for research applications. The core function of this product is to provide a reliable and standardized solution for researchers to conduct their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ab6556, by Thermo Fisher Scientific (1 mentions)
Irdye 680rd donkey anti rabbit, by LI COR (1 mentions)
Irdye 800cw donkey anti goat, by LI COR (1 mentions)
Newblot pvdf stripping buffer, by LI COR (1 mentions)
Irdye 800cw goat anti mouse, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Ab2787, by Abcam (1 mentions)
Ab13492, by Abcam (1 mentions)
Ab110271, by Abcam (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Odyssey dlx imager, by LI COR (1 mentions)
Irdye 680rd, by LI COR (1 mentions)
3 protocols using ab170325
1
Quantifying Protein Expression by Western Blot
2
Epitope Tagging of Yeast Proteins
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We inserted the gene sequences encoding 3 × HA tag at the 3΄ terminus of the PUT2, Pro1, Pro3, CAR1 and CAR2 genes, and the sequences encoding 13 × Myc tag at the 3΄ terminus of the MCH5 and PUT1 genes, in both D419A/ScΔleuS and WT/ScΔleuS cells by PCR-mediated homologous recombination (54 (link)). Correct integration was confirmed by PCR. After treatment with or without 0.5 mM Nva for 12 h, the yeast cells were harvested and resuspended in ice-cold lysis buffer containing 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100, 1 mM PMSF and 1 mM DTT, mixed with a few glass beads, vortexed rigorously for 30 min, then centrifuged at 10 000 × g for 15 min, 4°C. The supernatant was collected and proteins were detected by western blot. Anti-HA antibody was obtained from Sigma (H3663), anti-Myc antibody was from Proteintech (66004), anti-beta Actin antibody (ab170325), anti-Hsp70 antibody (ab2787) and anti-Hsp90 antibody (ab13492) were from Abcam.
3
Western Blot Analysis of Yeast Mitochondrial Protein
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Cells were grown as described for “Oxygen consumption assay”. 2 × 108 cells were resuspended in 0.8 ml of 200 mM NaOH and incubated at room temperature for 5 min. Cells were collected by centrifugation and the pellet was resuspended in 0.15 ml loading buffer and boiled for 5 min 10 μl of each sample was used for polyacrylamide gel electrophoresis (PAGE), followed by western blotting onto Immobilon PVDF membrane (0.2 μm) (Millipore). The resulting membranes were probed with antibodies specific for either yeast Cox2p (anti-MTCO2; Abcam ab110271) at a dilution of 1:500 or for yeast Act1p (anti-β-Actin; Abcam ab170325) at a dilution of 1:5000. Secondary antibodies were IRDye800CW and IRDye680RD (Licor). Proteins were visualized using Odyssey DLx imager (Licor) and signal intensity was analyzed using the provided software (Empiria Studio–Licor).
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