I would like to estimate total protein from crab muscle tissue samples and would like to know if a chilled centrifuge is necessary (my lab does not have one). I plan to use the Pierce* 660 nm Protein Assay in order to quantify the amount of protein.
It would depend on the length of time from start to finish - from when you kill the crab to dissecting out the muscle, to homogenizing the tissue, and centrifugation. Don't forget that a centrifuge can build up heat during the spin. How many rpm or g-forces, and for how long? From your post I assume that a crude homogenate is all you need. Is the centrifugation step just to remove large cellular and/or tissue debris? If it is a small footprint benchtop model centrifuge, you can always put the instrument into a cold room or even a refrigerator (but is there an electrical outlet inside?).
The length of time is a little variable but I can keep the samples on ice throughout the process. The current protocol I am using asks for 20 minutes at 15,000 gs. Yes all i need is the crude homogenate and the centrifugation step is just to remove tissue debris. I may be able to run a cord into a refrigerator- great idea.
I suggest the following: It may be that crab muscle has a lot of connective tissue. After you homogenize (BTW, are you using a Dounce-type homogenizer or a motorized bladed shredder-type?), pass the homogenate through several layers of wetted gauze. That will remove a lot of the gunk. 20 min @ 15K spin is standard for pelleting crude mitochondria. You don't need that. 600 to 1000 X g for 10 min is sufficient for a crude homogenate for total protein assay.
Dear Zaveta,
while dealing with the proteins, it is always important to avoid the protein from denaturation. so during homogenization and centrifugation it is important to maintain the temperature. for this purpose you can use ice for homogenization and temperature controlled centrifugation machine.
Good Luck
Dear Zaveta,
if you are measuring only total protein I think centrifugation temperature does not play such a crucial role; much more variability is induced by the choice of the general extraction and determination methods. I have used 20 min with 10,000 g as a standard centrifugation method (for enzymatic measurements WITH cooling).
I agree with Kari. Although the best practice would be to work on ice for most all assays, for total protein by the method of Pierce this will make little or no difference. The assay is not influenced by the conformation of your proteins, but rather by the amino acid composition, which is in total protein extracts of muscle of much less concern. Obviously temperature and timing of the assay itself is important.
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