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Myofibrillar extraction from mouse heart
Posted by: Raju (IP Hidden, Unregistered user, )
Date: February 16, 2005 06:42PM
Hi There,
Does any body have the protocol for the extraction of myofibrillar proteins from atria and ventricles of mouse heart? If so could you let me know. I tried a couple of times with the following protocol but no success. It would be grteful for me if could give me some tips or modifications in my protocol. 1) I took hearts and then rinsed in ice cold LSB (low salt buffer: 20mM KCl, 2mM K2HPO4; 1mM EGTA; pH 6.8) to remove blood. 2) Separated atria and ventricles and then weighed them separately. 3) Minced the heart tissue with razor blade and then homogenised in 1 ml LSB. 4) Spin down the sample at 5000 rpm for 10 min at 4 Celsius. 5) At the end of spin, supernatant was discarded and replaced with fresh LSB and then spun again at 5000 rpm for 10 min at 4 C. 6) Discraded the supernatant at the end of spin and resupended the pellet in HSB (high salt buffer: 40mM Na4P2O7; 1mM MgCl2; 1mM EGTA. pH 9.5) and then incubated for 30 min at 4C. 7) Spun down the sample at 13200 rpm for 15 min at 4C. 8) collected the supernatant which contains myofibrillar extract. At the end I measured the protein conc by Bradford then ran the samples on 10%SDS-PAGE gel. I was able to see only actin band (44 kDa) but not myosin band which I am expecting. It seems to be the protein degraded but I am not sure. If it the case is it recommendable to use any protein inhibitors in the LSB and HSB. It would be grateful for me if somebody can help in sorting out this problem. Thanks
Re: Myofibrillar extraction from mouse heart
Posted by: TR (IP Hidden, New member, 1)
Date: May 17, 2005 02:55PM
Hi: I am also looking for a protocol to extract myofibrillar proteins from mouse heart.
Did u have a working protocol now? thanks, TR
Re: Myofibrillar extraction from mouse heart
Posted by: Ref2Circ (IP Hidden, Unregistered user, )
Date: May 17, 2005 03:36PM
Circulation. 1999;99:505-510
Protein Kinase A (PKA)-Dependent Troponin-I Phosphorylation and PKA Regulatory Subunits Are Decreased in Human Dilated Cardiomyopathy Daniel R. Zakhary, BS; Christine S. Moravec, PhD; Robert W. Stewart, MD; Meredith Bond, PhD Preparation of Cardiac Myofibrils Samples of cardiac left ventricular free wall (5 g) from patients with end-stage (New York Heart Association class IV) idiopathic DCM and from NF donor hearts were homogenized in 5 vol of MOPS buffer (10 mmol/L, pH 7.4, 4°C) containing sucrose (290 mmol/L), NaN3 (3 mmol/L), DTT (1 mmol/L), NaF (20 mmol/L), pepstatin A (1 µmol/L), leupeptin (1 µmol/L), and PMSF (0.8 mmol/L) by use of three 20-second bursts and 40-second cooling intervals, position 6, with a Polytron homogenizer. This yielded total heart homogenate (THH). An equivalent volume of extraction buffer, 50 mmol/L Tris (pH 7.5) containing Triton X-100 (0.1%), NaF (20 mmol/L), DTT (0.05 mmol/L), MgCl2 (0.5 mmol/L), EDTA (0.125 mmol/L), antipain (5 µg/mL), leupeptin (10 µg/mL), pepstatin A (5 µg/mL), and PMSF (43 µg/mL), was added to the THH, and the mixture was centrifuged at 10 000g for 5 minutes at 4°C. The detergent-solubilized supernatant was set aside, and the pellet was resuspended in an equivalent volume of extraction buffer and washed twice again. The resultant myofibrillar fraction was resuspended in Ca2+-free extraction buffer and stored at -20°C.
Re: Myofibrillar extraction from mouse heart
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 17, 2005 04:49PM
yes you can use protease inhibitors such as PMSF, Pepstatin A and leupeptin to prevent the breakdown of protein. check the literature to see the recommended concentrations.
Re: Myofibrillar extraction from mouse heart
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 24, 2005 02:47PM
you may also want to try a protease inhibitor cocktail, instead of worrying about the concentrations you need to add of various inhibitors. we use one from Sigma for mammalian cells and tissues.
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