Protein Extraction and Immunoblotting from Frozen Brains

Dissected brains were immediately frozen in liquid nitrogen and stored at −80°C. For protein extraction, frozen brains were manually homogenized in Hunt buffer (20 mM Tris-HCl [pH 8.0], 100 mM sodium chloride, 1 mM EDTA, 0.5% NP-40) supplemented with protease inhibitor cocktail (Roche) and phenylmethylsulfonyl fluoride (PMSF) in a glass homogenizer. After full-speed centrifugation, the supernatant containing the soluble protein fraction was further used. Equal amounts of 20 to 30 μg of proteins were separated by SDS-PAGE (10% gels) and transferred onto nitrocellulose membranes (Protran; Whatman) according to standard protocols. For detection, the Enhanced Chemiluminescence kit (PerkinElmer) was used. HDAC activity assays were performed with brain protein extracts as previously described (9 (link)). Primary antibodies for immunoblotting were HDAC1 (10E2 or Sat13), HDAC2 (3F3), Sin3A (catalog number sc-994; Santa Cruz), CoREST (catalog number 07-455; Millipore), MTA1 (catalog number sc-9446; Santa Cruz), PKCδ (catalog number 610397; BD), and β-actin (catalog number A5316; Sigma) antibodies.

Free full text: Click here

Hagelkruys A., Mattes K., Moos V., Rennmayr M., Ringbauer M., Sawicka A, & Seiser C. (2016). Essential Nonredundant Function of the Catalytic Activity of Histone Deacetylase 2 in Mouse Development. Molecular and Cellular Biology, 36(3), 462-474.

Publication 2015

protease inhibitor cocktail Protran Enhanced Chemiluminescence kit Sin3A CoREST β-actin

Actin Antibodies Assays Brain Buffer Centrifugation Chemiluminescence Edta Frozen Gels Membranes Mta1 Nitrocellulose Nitrogen Np 40 Phenylmethylsulfonyl fluoride Protease inhibitor Protein Sds page Sodium chloride Tris

Corresponding Organization :

Other organizations : Max Perutz Labs, Medical University of Vienna, Vienna Biocenter

Top 5 similar protocols

Variable analysis

independent variables

Dissected brains were immediately frozen in liquid nitrogen and stored at −80°C

dependent variables

HDAC activity
Protein expression levels (HDAC1, HDAC2, Sin3A, CoREST, MTA1, PKCδ, β-actin)

control variables

Equal amounts of 20 to 30 μg of proteins were separated by SDS-PAGE (10% gels) and transferred onto nitrocellulose membranes
Hunt buffer (20 mM Tris-HCl [pH 8.0], 100 mM sodium chloride, 1 mM EDTA, 0.5% NP-40) supplemented with protease inhibitor cocktail (Roche) and phenylmethylsulfonyl fluoride (PMSF) for protein extraction

controls

Positive control: Not specified
Negative control: Not specified

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!