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Memcode staining

Manufactured by Thermo Fisher Scientific
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MemCode is a membrane staining solution that can be used to visualize and quantify protein in Western blot analysis. It provides a rapid and sensitive way to stain and detect membrane-bound proteins.

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Lab products found in correlation

Bca kit, by Merck Group (1 mentions) Cellytic mt lysis buffer, by Merck Group (1 mentions)

3 protocols using memcode staining

1

Profiling AKT1-mediated Cortactin Phosphorylation

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GST fusion proteins were generated in yeast or bacterial systems and purified with Glutathione-Sepharose beads. The GST-fusion proteins were pretreated with lambda phosphatase (NEB) at 30°C for 30 minutes and incubated at 60°C for another 30 minutes to inactivate any phosphatase activity. Pretreated GST-fusion proteins were mixed with 32P-ATP (250 µCi) and with or without 100 ng AKT1 for 30 minutes at 30°C. The samples were resolved by SDS-PAGE and the proteins transferred onto nitrocellulose membranes. Phosphorylated and total proteins were visualized by autoradiography or MemCode staining (Thermo), respectively. To examine AKT1-induced phosphorylation sites, cortactin-GST fusion proteins were incubated with 1 mM ATP in the presence of absence of AKT1 at 30°C for 30 minutes. The samples were then resolved by SDS-PAGE and visualized by coomassie blue staining. Gel bands were excised and digested with tryspin. Tryptic peptides were extracted and analyzed on an LTQ-Orbitrap Elite mass spectrometer. Mass spectrometry data were searched against Human RefSeq with Sequest and Mascot algorithms.
Wu X., Renuse S., Sahasrabuddhe N.A., Zahari M.S., Chaerkady R., Kim M.S., Nirujogi R.S., Mohseni M., Kumar P., Raju R., Zhong J., Yang J., Neiswinger J., Jeong J.S., Newman R., Powers M.A., Somani B.L., Gabrielson E., Sukumar S., Stearns V., Qian J., Zhu H., Vogelstein B., Park B.H, & Pandey A. (2014). Activation of diverse signaling pathways by oncogenic PIK3CA mutations. Nature communications, 5, 4961.
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2

Profiling AKT1-mediated Cortactin Phosphorylation

Check if the same lab product or an alternative is used in the 5 most similar protocols
GST fusion proteins were generated in yeast or bacterial systems and purified with Glutathione-Sepharose beads. The GST-fusion proteins were pretreated with lambda phosphatase (NEB) at 30°C for 30 minutes and incubated at 60°C for another 30 minutes to inactivate any phosphatase activity. Pretreated GST-fusion proteins were mixed with 32P-ATP (250 µCi) and with or without 100 ng AKT1 for 30 minutes at 30°C. The samples were resolved by SDS-PAGE and the proteins transferred onto nitrocellulose membranes. Phosphorylated and total proteins were visualized by autoradiography or MemCode staining (Thermo), respectively. To examine AKT1-induced phosphorylation sites, cortactin-GST fusion proteins were incubated with 1 mM ATP in the presence of absence of AKT1 at 30°C for 30 minutes. The samples were then resolved by SDS-PAGE and visualized by coomassie blue staining. Gel bands were excised and digested with tryspin. Tryptic peptides were extracted and analyzed on an LTQ-Orbitrap Elite mass spectrometer. Mass spectrometry data were searched against Human RefSeq with Sequest and Mascot algorithms.
Wu X., Renuse S., Sahasrabuddhe N.A., Zahari M.S., Chaerkady R., Kim M.S., Nirujogi R.S., Mohseni M., Kumar P., Raju R., Zhong J., Yang J., Neiswinger J., Jeong J.S., Newman R., Powers M.A., Somani B.L., Gabrielson E., Sukumar S., Stearns V., Qian J., Zhu H., Vogelstein B., Park B.H, & Pandey A. (2014). Activation of diverse signaling pathways by oncogenic PIK3CA mutations. Nature communications, 5, 4961.
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3

Protein Expression Analysis Protocol

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Protein was isolated using CelLytic MT lysis buffer (Sigma Chemicals, St. Louis, MO) and quantified by using a bicinchoninic acid (BCA) kit (Sigma Chemicals, St. Louis, MO). Protein (50μg from cell/tissue lysates) was separated by SDS-PAGE, transferred to nitrocellulose, and incubated with antibodies prepared with fish gelatin blocking buffer (0.5% fish gelatin, 1X PBS, 1 mg/ml casein and 15 mM NaN3). IgG-congugated AlexFluor fluorescent secondary antibodies (Invitrogen) were used with the Li-COR (Li-COR Biosciences) to visualize bands. Protein expression was normalized to total protein as determined by MemCode staining (Thermo Scientific). The primary CrAT antibody was a generous gift from the laboratory of Dr. Fausto Hegardt.
Seiler S.E., Koves T.R., Gooding J.R., Wong K.E., Stevens R.S., Ilkayeva O.R., Wittmann A.H., DeBalsi K.L., Davies M.N., Lindeboom L., Schrauwen P., Schrauwen-Hinderling V.B, & Muoio D.M. (2015). Carnitine Acetyltransferase Mitigates Metabolic Inertia and Muscle Fatigue During Exercise. Cell metabolism, 22(1), 65-76.
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