Protein lysates were prepared from transfected cells using a buffer containing 0.05 M Tris-HCl at pH 8.0, 0.15 M NaCl, 5.0 mM EDTA, 1% NP-40 and a protease inhibitor cocktail at 4°C. 50µg of lysate was loaded per lane on a 10% sodium dodecyl sulfate-polyacrylamide gel in loading buffer containing 7.7mg/ml dithiothreitol (DTT). Detection for Western blot was performed with primary antibodies to SEC24D (ab191566; Abcam, Cambridge, MA, USA) at 1:2000, SEC23A (ab179811; Abcam, Cambridge, MA, USA) at 1:100, OASIS (ab33051; Abcam, Cambridge, MA, USA) at 1:1000, and GAPDH (ab37168; Abcam, Cambridge, MA, USA) at 1:2000. The secondary antibodies Goat Anti-Rabbit IgG H&L (ab6721; Abcam, Cambridge, MA, USA) and Goat Anti-Mouse Secondary IgG (TA130004; OriGene, Rockville, MD, USA) were used at a 1:20 000 dilution. Detection was achieved using Amersham ECL Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA). Quantitation of blot signals was achieved using ImageJ.12 Adjusted signal intensities were obtained by normalizing to GAPDH signal to control for loading differences between lanes.
Ab33051
Manufactured by Abcam
Sourced in United States
Ab33051 is a lab equipment product offered by Abcam. It serves as a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg h l, by Abcam (2 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Ab37168, by Abcam (2 mentions)
Ab191566, by Abcam (2 mentions)
Ab179811, by Abcam (2 mentions)
Goat anti mouse secondary igg ta130004, by OriGene (2 mentions)
Amersham ecl western blotting, by Cytiva (2 mentions)
Horseradish peroxidase hrp labeled secondary antibody, by Merck Group (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
A5441, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
3 protocols using ab33051
1
Western Blot Characterization of COPII Proteins
2
Western Blot Characterization of COPII Proteins
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Protein lysates were prepared from transfected cells using a buffer containing 0.05 M Tris-HCl at pH 8.0, 0.15 M NaCl, 5.0 mM EDTA, 1% NP-40 and a protease inhibitor cocktail at 4°C. 50µg of lysate was loaded per lane on a 10% sodium dodecyl sulfate-polyacrylamide gel in loading buffer containing 7.7mg/ml dithiothreitol (DTT). Detection for Western blot was performed with primary antibodies to SEC24D (ab191566; Abcam, Cambridge, MA, USA) at 1:2000, SEC23A (ab179811; Abcam, Cambridge, MA, USA) at 1:100, OASIS (ab33051; Abcam, Cambridge, MA, USA) at 1:1000, and GAPDH (ab37168; Abcam, Cambridge, MA, USA) at 1:2000. The secondary antibodies Goat Anti-Rabbit IgG H&L (ab6721; Abcam, Cambridge, MA, USA) and Goat Anti-Mouse Secondary IgG (TA130004; OriGene, Rockville, MD, USA) were used at a 1:20 000 dilution. Detection was achieved using Amersham ECL Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA). Quantitation of blot signals was achieved using ImageJ.12 Adjusted signal intensities were obtained by normalizing to GAPDH signal to control for loading differences between lanes.
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from cells using lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton-X 100, 1% DTT, and 1% protease inhibitor cocktail (Roche, Basel, Switzerland). Equal amounts of protein extracts (40 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. Membranes were blocked with 5% w/v non-fat dry milk dissolved in Tris buffered saline plus Tween-20 (TBS-T; 0.1% Tween-20; pH 8.3) at room temperature for 1 h, then incubated with primary antibodies at 4 °C overnight. The following antibodies were used for Western blotting: mouse monoclonal anti-β-actin (A5441, Sigma-Aldrich, MO, USA), rabbit polyclonal anti-CREB3L1 (ab33051, Abcam, MA, USA), and rabbit polyclonal anti-FGFBP1 (sc-292235, Santa Cruz Biotechnology, TX, USA). After washing with TBS-T, membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (Sigma, USA) for 1 h at room temperature. Immunobands were visualized using enhanced chemiluminescence (ECL) kit (GE Healthcare, Waukesha, WI, USA) according to manufacture’s instructions.
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