I freeze fungal mycelia of mushroom. In SDS-PAGE of total protein ,If I freeze mycelium I can used this sample?
How should I prepare mycelium for electrophoresis? How should I make it for electrophoresis if I freeze mycelium?
plaese help me!!!
In principle, you can use the same method that you use for fresh tissue. Heating in SDS sample buffer will unfold proteins anyway, so any cold denaturing that may occur during storage should not influence the result.
One caveat: freezing will have ruptured intracellular membranes like lysosomes, and released proteolytic enzymes from them.
I agree with Engelbert, frozen samples should be fine; just heat/boil your samples in SDS sample buffer. One thing that you could do in the future is to quick freeze your mycelium samples in liquid nitrogen or dry ice/ethanol and store the frozen samples at -80C degrees to best preserve the samples if you like to store the samples long term.
Hua Xiao
Engelbert Buxbaum
Thanks for your attention.
Your description was very useful for understanding the subject
Do you have any protocols in this regard?
I have both fresh tissue and freez-dry sample
Which one of them is better to use?
It's not quite clear whether your sample is freeze dried or just frozen from your question, however, either way I would recommend the following protocol. This protocol works great for fresh mushroom tissue also.
1. Take a small (~100 mL) mortar and pestle and fill it with liquid nitrogen with the pestle sitting inside the mortar. Drop the frozen/freeze dried tissue in.
2. Allow the liquid nitrogen to evaporate, the mortar/pestle and tissue are now all at liquid nitrogen temperature.
3. Grind the tissue to a very fine powder
4. Add the powdered tissue to about 5 - 10 volumes of Laemmli buffer (50 mM Tris-HCl pH 6.8, 2 % SDS, 50 mM 2-mercaptoethanol, 10% glycerol)
5. Allow the mixture to warm to room temperature and mix (vortex) often
6. Sit at room temperature 20 min, with frequent vortexing
7. Heat at 95 C for 10 min then chill briefly on ice
8. Centrifuge at 15000 X G for 20 min
9. Remove supernatant
Supernatant protein concentration can be determined using the absorbance at A280 and A260 using the following formula: concentration (mg/mL) = (1.55 X A280) - (0.76 X A260). This will give a rough estimate of the amount of protein. It's not super accurate if you want to know how much protein is actually present with great accuracy but it will be completely fine for purposes of equal loading of samples.
When you are ready to load your samples, simply add 1 uL/100 uL of 1% w/v bromophenol blue to the protein sample and load the required amount on the gel. There is no need for further heating. Samples can be stored at -20 for years without degradation.
Keep in mind this protocol is only good for protein extraction for SDS-PAGE. If you need to extract protein from frozen samples under non-denaturing conditions from fungal material I have protocols for that too so just post a reply if you need more info.
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