Immunoblotting Analysis of Signaling Proteins in MCF7 Cells

Adherent MCF7 cells were scraped, and centrifuged with sterile PBS for collection and resuspended in RIPA buffer followed by pulse sonication. Westerns were performed as described [14 (link)]. Antibodies against the following proteins were used, typically at 1:2000 dilution: MDR-1 (Sigma), BCRP (Santa Cruz Biotechnology; SCBt), HIF1α (Abcam), S6K total (SCBt), S6K^S411phos (SCBt), p53 (SCBt), p53^S392phos (Abcam), TFPI1 (Abcam), AMPKα1/2 total (SCBt), AMPKα1^T183/2^T172phos (Abcam), AKT total (SCBt), AKT^S473phos (SCBt), PARP (Sigma), ERα (SCBt), histone H3 total (Millipore), H3^K9Ac (Millipore), H2B total (Abcam), H4^K12Ac (Abcam), NFκB (SCBt), NREL (SCBt), tubulin (Sigma), actin (Sigma), and GAPDH (Millipore). Luminescence was captured on film (Kodak) with subsequent chemical development. Collection and semiquantitation of Western blots densitometry was done using ImageJ Version 1.51 from scans of the original film.

Free full text: Click here

Davies G., Lobanova L., Dawicki W., Groot G., Gordon J.R., Bowen M., Harkness T, & Arnason T. (2017). Metformin inhibits the development, and promotes the resensitization, of treatment-resistant breast cancer. PLoS ONE, 12(12), e0187191.

Publication 2017

MDR-1 HIF1α H3K9Ac H4K12Ac tubulin actin GAPDH

Actin Antibodies Buffer Densitometry Dilution Gapdh Hif1α Histone h3 Luminescence Mcf7 cells Nfκb Proteins Pulse Ripa Scans Sterile Tubulin Western blots

Corresponding Organization : University of Saskatchewan

Top 5 similar protocols

Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of MDR-1, BCRP, HIF1α, S6K total, S6K^S411phos^, p53, p53^S392phos^, TFPI1, AMPKα1/2 total, AMPKα1^T183^/2^T172phos^, AKT total, AKT^S473phos^, PARP, ERα, histone H3 total, H3^K9Ac^, H2B total, H4^K12Ac^, NFκB, NREL, tubulin, actin, and GAPDH

control variables

Adherent MCF7 cells were used
Cells were scraped, centrifuged with sterile PBS for collection, and resuspended in RIPA buffer followed by pulse sonication
Western blots were performed as described in the reference [14]
Antibodies were used typically at 1:2000 dilution

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!