Protein Extraction from Brain Tissue

The harvested brain tissues were homogenized on ice using an immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40) plus protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A). The lysates were collected, centrifuged at 12,000 rpm for 10 min, and quantified for total proteins using a bicinchoninic acid protein assay kit (Pierce, Iselin, NJ).

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Zhang Y., Zhen Y., Dong Y., Xu Z., Yue Y., Golde T.E., Tanzi R.E., Moir R.D, & Xie Z. (2011). Anesthetic Propofol Attenuates the Isoflurane-Induced Caspase-3 Activation and Aβ Oligomerization. PLoS ONE, 6(11), e27019.

Publication 2011

Aprotinin Assay Bicinchoninic acid Brain Buffer Edta Immunoprecipitation Leupeptin Nacl Nonidet p 40 Pepstatin Protease inhibitors Protein Tissues Tris

Corresponding Organization : University of Florida

Top 5 similar protocols

Protocol cited in 2 other protocols

Variable analysis

independent variables

Homogenization of harvested brain tissues

dependent variables

Total protein quantification

control variables

Use of immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40)
Addition of protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A)
Centrifugation at 12,000 rpm for 10 minutes

controls

No positive or negative controls were explicitly mentioned.

Annotations

Based on most similar protocols

The protocols use similar buffers and centrifugation conditions to lyse and extract proteins from brain tissue samples. The input protocol and Protocols 1-3 use an immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40) plus protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A) for homogenization. (Input protocol, Protocols 1-3)

The lysates are then centrifuged at 12,000-13,000 rpm for 10-15 minutes to remove insoluble debris. (Input protocol, Protocols 1-3)

The total protein concentration in the lysates is quantified using a bicinchoninic acid (BCA) protein assay kit. (Input protocol, Protocols 1-3)

Protocol 4 uses a different lysis buffer containing 1% Triton X-100, 1 mM dithiothreitol (DTT), and protease inhibitor cocktail. The lysates are then centrifuged at 15,000 × g for 5 minutes. Protein concentration is determined by a detergent compatible (DC) protein assay.

Protocol 5 uses RIPA buffer containing protease inhibitors (aprotinin, leupeptin, phenylmethylsulfonyl fluoride) for homogenization. The lysates are centrifuged at 13,000 rpm for 15 minutes, and the protein concentration is determined by a Bio-Rad DC protein assay kit.

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