Mouse monoclonal anti ha antibody

Yeast total extracts were prepared following trichloroacetic acid
precipitation method (Knop et
al., 1999). 5 OD of cells were resuspended with 1150 μL
alkaline lysis buffer (0.24 N NaOH, 1% β-mercaptoethanol, 1X EDTA-free
protease inhibitor cocktail (Roche), 1 mM EDTA, 1 mM PMSF, 5 μM Pepstatin
A, 10 μM Leupeptin, 0.2 mM sodium orthovanadate, 10 mM
β-glycerolphosphate, 10 mM NaF and 10 mM NaN₃). After
incubation on ice for 15 min, 150 μL of 55% TCA was added to precipitate
protein for 10 min and followed by 16000×g centrifugation for 10 min at
4°C. The pellet was resuspended in 250 μL HU buffer (8 M urea, 5%
SDS, 200 mM Tris-HCl pH 6.8, 1 mM EDTA, 5% β-mercaptoethanol and 1%
Bromophenol blue) and incubated in 65°C for 10 min, followed by
16000×g centrifugation for 5 min at RT. The supernatant was subjected to
SDS-PAGE for protein analysis.
Antibodies: mouse anti-FLAG M2 antibody (Sigma),
mouse anti-Por1 monoclonal antibody (Invitrogen), mouse anti-Cox2 monoclonal
antibody (Invitrogen), mouse anti-Pgk1 monoclonal antibody (Invitrogen), mouse
anti-GFP monoclonal antibody (Roche, clone 7.1 and 13.1), and mouse anti-HA
monoclonal antibody (Roche, clone 12CA5).