SOP_LEP-URBO Protein extraction from animal tissues with RIPA buffer

Contributed by: Dietmar Kültz, modified by F. Silvestre

Latest version: 19 august 2008
This procedure will be performed as previously established (Kültz 1996; Kültz and Somero 1996; Valkova et al.
2005).

I. Preparation of total protein from animal tissues

1. Dissect the tissue, cover in labeled aluminum foil, and snap-freeze immediately in liquid nitrogen.
2. Store tissues at - 80°C (avoid freezing-thawing cycles)
3. Weigh frozen tissue, and add RIPA buffer at a ratio of 0.4 ml per 0.1 g tissue (1:4 w:v).
4. Homogenize on ice using a tight-fitting glass homogenizer and transfer homogenate into a 2 ml siliconized
tube (Fisher cat. # 02-681-332).
5. Incubate on ice for 10 min to maximize protein solubilization.
6. Centrifuge at 19,000g and 4°C for 10 min.
7. Save the supernatant into a clean 2 ml siliconized tube (save 20 µl in a separate tube for protein
determination). Ex: for a sturgeon larvae of about 40 mg, we store about 5 X 20 µL supernatant).
8. Snap-freeze the sample in liquid nitrogen.
8. Store at -80°C or use immediately.
Composition of RIPA buffer:
- RIPA buffer stock sol.(stock at RT): 50 mM Tris-HCl, pH 7.4 - 7.6 (dil. 1M stock); 150 mM NaCl (8.8g/ L).
- Add 1% NP-40; 1% Triton X-100; 1% CHAPS; 1 mini tablet Complete (Roche protease inhibitors), 100 µl
NaF stock sol. (200 mM, RT) (= phosphatase inhibitor), 100 µl activated Na3VO4 stock sol. (200 mM, 4°C,
stored in 50 µl aliquots) to 10 ml RIPA buffer stock solution.

Reference List

Kültz,D. 1996. Plasticity and stressor specificity of osmotic and heat shock responses of Gillichthys mirabilis gill
cells. Am J Physiol 271: C1181-C1193.

Kültz,D. and Somero,G.N. 1996. Differences in protein patterns of gill epithelial cells of the fish Gillichthys
mirabilis after osmotic and thermal acclimation. J Comp Physiol [B] 166: 88-100.

Valkova,N., Yunis,R., Mak,S.K., Kang,K., and Kültz,D. 2005. Nek8 Mutation Causes Overexpression of Galectin-
1, Sorcin, and Vimentin and Accumulation of the Major Urinary Protein in Renal Cysts of jck Mice. Mol. Cell
Proteomics. 4: 1009-1018.