Jump to navigationJump to search
Back to Lidstrom:Protocols
- sample should be on ice during sonication and between sonication rounds to preserve the activities of the proteins you are extracting.
- There is a power setting (0-99) that will affect how effectively each pules-second is, but also how hot the sample will get from each pulse.
How should I do the pulses?
- The design criteria for the pulsing are: (a) enough seconds of sonication to actually break open cells (b) keeping the samples as cool as possible while this happens.
- Ceci advised that keeping the temperature below 10oC is good enough.
- culture volume
- viscosity (probably)
- power setting
- amount & frequency of pulse seconds
- In principle for a given sample size there are frequencies at which you can pulse and wait that will not lead to net increase in temperature (read after sonication) over time.
- You can measure the temperature of the samples with a heat-block style stick/alcohol thermometer.
- (wipe clean between samples!)
- TIP #1: If you want to know whether your sample is above or below 10oC, it is best to start with the thermometer close to 10oC so you can see whether the reading goes up or down after insertion into the sample. If your thermometer is too hot or too cold than the time it takes for the sample to reach equilibration with the thermometer has allowed for a lot of time for the sample to be cooled by the ice it should be sitting in. Also, if the thermometer is too hot, you are adding heat to your sample unnecessarily.
- TIP #2: You can test the protocol you want to use on a volume of buffer equivalent to the samples you will be sonicating to test how hot your cycling protocol gets the samples.
How many pulse seconds should be applied per sample?
- This is actually a tricky question that requires better data than anyone in our lab seems to have. The best test would be to have many tubes of biomass, test different amount of pulse seconds, then measure the protein concentration with BCA to see if there is an asymptotic relationship between the total number of seconds and the resulting soluble protein concentration.
- Note: the amount of pulse seconds required likely depends on the cell concentration and the strain you are using.
What volume of cells should I use?
- ~1/2 a mL is suitable for our sonicator tip size.
How deep should the shaft be?
- You don't want it to foam, so it should be near but not touching the bottom of the eppendorf tube.
Ceci's AM1 protocol 5/2013
This protocol is for the sonicator in the glove box, which will behave differently than the one the rest of us use.
- One "hit" = 45 seconds intermitent pulses (one second pulse, two second off more or less)
- Power setting = 6 (out of 10?)
- Wait around 4 min in between "hits"
- Always measure temp with the thermometer after each "hit."
- Do 6 hits total.
- Results: My concentration, determined by BCA, was comparable to our method using the french press.
- Note: For AM1, the french press is faster and more efficient. This protocol was developed because she had to do anaerobic work in the anaerobic chamber, where the french press can't be used.
Janet's Tests 5/2013
- Janet had programmed the sonicator to pulse at 99% of max power for 1 sec, and not pulse for 30 seconds and that was sufficient to not heat up samples w/ volume = 1/2 mL.
- On for one sec and off for <10 sec definitely leads to excessive heating at this power setting and liquid volume