Protein lysates were prepared by lysing cells in RIPA buffer containing protease inhibitor cocktail. The protein concentration was determined using the BCA protein assay (Thermo Scientific), and 30 μg of proteins were separated by SDS-PAGE and transferred to a 0.2 mm nitrocellulose membrane. The membranes were blocked by tris-buffered saline (TBS; 0.2 M Tris base, 1.5 M NaCl), 0.1% Tween-20, and 5% ECL blocking reagent (GE Healthcare) for one hour, then incubated with the primary antibody, GLUT1 (Abcam, ab15309), GLUT4 (Santa Cruz, sc-53566), Akt (Cell Signaling, #9272), P-Akt (Cell Signaling, #9271), heavy chain myosin (Abcam, ab124205) or glycogen synthase (Cell Signaling, #3893). Then, the membranes were incubated with HRP-conjugated secondary antibody (Abcam) diluted in TBS with 0.1% Tween-20. After the addition of the HRP substrate, the chemiluminescent signal was detected using a Gel and Blot Imaging System (Azure Biosystems). The same membrane was stripped and reused for detection of β-actin (Abcam, ab8229) or α-Tubulin (Sigma, #T6074) as a loading control, following the same protocol as above.
Ab8229
Manufactured by Merck Group
Ab8229 is a laboratory equipment product from Merck Group. It is designed for general use in research and scientific applications. The core function of Ab8229 is to facilitate various laboratory tasks and procedures. Detailed specifications and intended use are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Glycogen synthase, by Cell Signaling Technology (2 mentions)
Hrp conjugated secondary antibody, by Abcam (2 mentions)
Gel and blot imaging system, by Azure Biosystems (2 mentions)
α tubulin, by Merck Group (2 mentions)
Ecl blocking reagent, by GE Healthcare (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
2 protocols using ab8229
1
Western Blot Analysis of Glucose Transporters
2
Western Blot Analysis of Glucose Transporters
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Protein lysates were prepared by lysing cells in RIPA buffer containing protease inhibitor cocktail. The protein concentration was determined using the BCA protein assay (Thermo Scientific), and 30 μg of proteins were separated by SDS-PAGE and transferred to a 0.2 mm nitrocellulose membrane. The membranes were blocked by tris-buffered saline (TBS; 0.2 M Tris base, 1.5 M NaCl), 0.1% Tween-20, and 5% ECL blocking reagent (GE Healthcare) for one hour, then incubated with the primary antibody, GLUT1 (Abcam, ab15309), GLUT4 (Santa Cruz, sc-53566), Akt (Cell Signaling, #9272), P-Akt (Cell Signaling, #9271), heavy chain myosin (Abcam, ab124205) or glycogen synthase (Cell Signaling, #3893). Then, the membranes were incubated with HRP-conjugated secondary antibody (Abcam) diluted in TBS with 0.1% Tween-20. After the addition of the HRP substrate, the chemiluminescent signal was detected using a Gel and Blot Imaging System (Azure Biosystems). The same membrane was stripped and reused for detection of β-actin (Abcam, ab8229) or α-Tubulin (Sigma, #T6074) as a loading control, following the same protocol as above.
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