Femur Decalcification and Protein Extraction

The mouse femurs were dissected and decalcified by EDTA before protein extraction [14 (link)]. The decalcified bone samples were chopped into small pieces and homogenized with TissueLyser 5mm stainless steel beads (Qiagen, Redwood City, CA) in the RIPA lysis buffer containing the proteinase inhibitor cocktail (ThermoFisher Scientific, Waltham, MA). Protein concentrations were measured with the BCA protein reagent (ThermoFisher Scientific). The p65 (D14E12) and phospho-p65 (93H1) were stained by the antibodies from Cell Signaling Technology (Danvers, MA). The horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) was used and the signals were detected by adding the enhanced chemiluminescence western blotting detection reagents (ThermoFisher Scientific). The quantification of western blotting results was done by Image Lab statistic software (Bio-Rad). The images were quantified by using ImageJ.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Lin T., Pajarinen J., Kohno Y., Nabeshima A., Lu L., Nathan K., Yao Z., Wu J.Y, & Goodman S. (2019). Increased NF-kB activity in osteoprogenitor-lineage cells impairs the balance of bone versus fat in the marrow of skeletally mature mice. Regenerative engineering and translational medicine, 6, 69-77.

Publication 2019

proteinase inhibitor cocktail BCA protein reagent horseradish peroxidase-conjugated secondary antibody enhanced chemiluminescence western blotting detection reagents

Antibodies Antibody Bone Buffer Chemiluminescence Edta Femurs Horseradish peroxidase Mouse Protein Proteinase inhibitor Redwood Ripa Stainless steel

Corresponding Organization : Stanford University

Top 5 similar protocols

Variable analysis

independent variables

EDTA decalcification of mouse femurs

dependent variables

Protein extraction
Protein concentration
P65 and phospho-p65 protein levels

control variables

Proteinase inhibitor cocktail
RIPA lysis buffer

controls

Positive control: None mentioned
Negative control: None 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!