PROTOCOL (For Cells)
1. Preparation of cells.
Note : After preparation of adherent cell or suspension cell in 50ml tube, centrifuge at 2,000- 3,000rpm for 5 min. Then wash cells with PBS/DPBS (optional). After washing, count cells and use approximately 5x 106 cells.
2. Harvest the cell pellet by centrifuge at 13,000 rpm for 10 -20 seconds.
Note : After centrifugation, remove the remnant using a pipette.
3. Resuspend cells in 400 ㎕ PRO-PREP TM solution, and mix well.
Note : Depending on tissue types, one can vary volume of PRO-PREP TM solution. Generally add 5x106 cell per 400㎕, but determine the optimal amount of solution according to cell size. Also, pipette carefu lly as the addition of PRO-PREP TM solution can produce bubbles.
4. Induce cell lysis by incubation for 10 -20 min on ice or freezer at –20℃.
Note : PRO-PREP TM solution don’t freeze at -20℃, and it can be stabilize protein refraining protein degradation with protease inhibitor. Before incubating, it can also increase cell lysis using a syringe(opti onal). At this time, there appears bubbles, yet doesn ’ t need to care because they disappear during centrifuging or incubation.
5. Centrifuge at 13,000rpm (4℃) for 5minutes, and transfer supernatant to a fresh 1.5ml tube.
6. Measure of protein concentration.
Note : When measuring protein concentration by Bradford’ method etc., PRO-PREPTM solution is made to have no absorbable hindrance, and so can decline an absorbable error.
Which step can I store the solution in a protocol I used?step 4 or step 5?
I would suggest to store at step 5 when you have taken out your lysate.
I would suggest to store at step 5 when you have taken out your lysate.
You can store after adding cell lysis buffer at -80C. Whenever you ready for protein quantification, thaw the samples on ice, sonicate for 5-10 seconds, centrifuge at 13,000 rpm (4C) for 5 min then collect the supernatant. Samples are ready for quantification.
I also think is better to store at -20ºC at step 5 when you have taken your lysate. But it would be also possible to add the lysis buffer and then store the sample at -80ºC.
So, yo can choose, but whatever you choose I will always do it the same way to be able to compare between samples and experiments.
Check the instructions on the Pro-Prep before putting at -80 to be sure it is compatible. It seems like the point of using this solution is to avoid water crystallization and degradation of protein by freeze-thaw. Spin down the cell lysate and recover your supernatant, then store as per Pro-Prep's instructions (whether -20 or -80). By this logic, there is no freeze-thaw to worry about so you can determine concentration at any time later.
Either after step 2 before you add the Pro-prep and then thaw on ice to continue or, as people have mentioned, after step 5 when when you have collected your lysate.
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