Liver caspase activity was measured as described (Lawson et al. 1999 (link)). In brief, frozen liver tissue was homogenized in 25 mM HEPES buffer containing 5 mM EDTA, 2 mM DTT and 0.1% CHAPS, and then centrifuged to get the homogenate. A fluorogenic substrate (Ac-DEVD-AFC, Enzo Life Sciences, Plymouth Meeting, PA) was added to the homogenate and fluorescence was measured with or without the presence of pan-caspase inhibitor (z-VAD-fmk, Enzo). Results are expressed as RFU per unit time per mg protein concentration. Western blotting was performed as described (Bajt et al. 2000 (link)) using a rabbit anti-caspase 3 antibody and a rabbit anti-beta-actin antibody (Cell Signaling Technology, Danvers, MA), and an anti-RIP3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The proteins were visualized using a goat anti-rabbit HRP conjugated antibody (Santa Cruz Biotechnology Santa Cruz, CA).
Rabbit anti beta actin antibody
Manufactured by Cell Signaling Technology
Sourced in Canada
Rabbit anti-beta-actin antibody is a primary antibody that specifically binds to the beta-actin protein. Beta-actin is a commonly used housekeeping protein for western blotting and immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Gradient acrylamide tris acetate gel, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (2 mentions)
Sc 1004, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Ecl prime, by GE Healthcare (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Hrp conjugated goat anti rabbit antibody, by Santa Cruz Biotechnology (1 mentions)
Ac devd afc, by Enzo Life Sciences (1 mentions)
Anti rip3 antibody, by Santa Cruz Biotechnology (1 mentions)
Z vad fmk, by Enzo Life Sciences (1 mentions)
Irdye 800cw donkey anti rabbit, by LI COR (1 mentions)
Vi cell xr system, by Beckman Coulter (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
4 protocols using rabbit anti beta actin antibody
1
Liver Caspase Activity Assay
2
Quantifying FVIII Protein Expression
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Cell number and viability were measured using a Vicell XR system (Beckman Coulter). Metabolites were measured using a BioProfile 100 Plus (Nova Biopmedical). The activity of FVIII was measured as chromogenic activity using an in-house version of the Coatest SP (Chromogenix, Instrumentation Laboratory, Milano, Italy) [26] . Protein lysates for Western blotting were prepared by spinning down 10e6 cells and resuspending the pellet in 200 μl of Mammalian Protein Extraction Reagent (M-PER) (Thermo) following manufacturer's instructions. 30 μg of each sample was used for Western blotting. Gels were submitted to western blotting using Novex/NuPage blotting system (Invitrogen). Primary antibodies used were 1:500 dilution of sheep polyclonal anti-human factor VIII (CL20035AP, Cedarlane labs, Burlington, ON, Canada) and 1:1000 dilution of rabbit anti-beta-actin antibody (Cell Signaling Technology, Danvers, Ma, USA). Secondary antibodies used were 1:20000 dilution of Donkey anti-Rabbit IRDye® 800 CW (LI-COR® Biosciences, Lincoln, Ne, USA) and 1:10000 dilution of Alexa Fluor® 680 donkey anti-sheep IgG (Invitrogen, Carlsbad, Ca, USA). The ladders used were Full Range Rainbow™ recombinant protein molecular weight marker (Ge lifescience, Piscataway, NJ, USA).
3
Quantifying Transfected Protein Expression
4
Quantifying Transfected Protein Expression
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3134 cells were transiently co-transfected with Halo-GR and GFP-RPB1 using jetPRIME (Plyplus transfection) according to the manufacturer’s instructions. Note that we changed transfection reagents from the SMT experiments, since virtually all cells were transfected with jetPRIME, enabling a fair comparison of the levels of transfected vs. endogenous proteins. By examining the fluorescence signal of GFP-RPB1, we confirmed that expression levels within individual transfected cells were similar between jetPRIME and Lipofectamine LTX.
Transfected cells were lysed in RIPA buffer with cOmplete Protease Inhibitor (Roche) and the protein concentration was measured using a Bradford assay (Bio-Rad). 10 µg of total proteins were loaded on a 3–8% gradient acrylamide Tris-Acetate gel (Invitrogen) and run for 1 hour at 100V. Proteins were then transferred to a PVDF membrane (Invitrogen) and stained overnight at 4°C with either anti-GR rabbit antibody (1:5000, sc-1004, Santa Cruz) or anti-beta-actin rabbit antibody (1:5000, 13E5, Cell Signaling). HRP-Goat anti-rabbit IgG (1:5000, Invitrogen) was used as a second antibody. The protein of interest was detected using ECL Prime (GE Healthcare).
Transfected cells were lysed in RIPA buffer with cOmplete Protease Inhibitor (Roche) and the protein concentration was measured using a Bradford assay (Bio-Rad). 10 µg of total proteins were loaded on a 3–8% gradient acrylamide Tris-Acetate gel (Invitrogen) and run for 1 hour at 100V. Proteins were then transferred to a PVDF membrane (Invitrogen) and stained overnight at 4°C with either anti-GR rabbit antibody (1:5000, sc-1004, Santa Cruz) or anti-beta-actin rabbit antibody (1:5000, 13E5, Cell Signaling). HRP-Goat anti-rabbit IgG (1:5000, Invitrogen) was used as a second antibody. The protein of interest was detected using ECL Prime (GE Healthcare).
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