Lysis buffer used to get cell lysates need to be sterilised? - (Mar/13/2008 )

Hi, do we need to sterilise the lysis buffer pH 6.8 (containing 0.1% Triton X-100) for cell lysis purpose?

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Hey,

I don`t know whether you have to do it but I do sterile filter it (and most of the my stock solutions/buffers etc.). I found out that I can keep them longer without any growth in.

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So, u store the stock solutions/buffers in the fridge or just put in room temperature? If just use to lyse the cells and release the enzymes, then add in substrate, incubate for 3 hrs and read absorbance value, do we still need to sterilise the lysis buffer?

If want to sterilise, can I choose to autoclave the buffer solutions?

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You don't neccessarily need the buffer to be sterile but as zek pointed out, you don't want growth. If you make up the buffer fresh, you don't need to worry about sterilizing. However, if you want to store your buffers long term you need to prevent growth. I always sterile filter because this also removes precipitants/fibers/aggregates which autoclaving will not. I suppose you could autoclave your buffer but be sure you add the detergent afterward! I also always store my buffers at 4degrees. Again, I'm not sure if this is absolutely neccessary but you always want to use cold buffers/wash solutions when lysing cells and this way my buffers are always cold. I also keep a bottle of pbs in the fridge for washing the cells.

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It depends on your solutions. If you have contamination, it will also grow in the fridge. It just takes longer than at RT. I usually prepare those kind of buffers fresh, then I do not sterile filter it (anyway, the stocks are sterile filtered). If I keep that buffer for a long time, I do sterile filter.



I prefer sterile filtering because in our lab we do not often use the autoclave machine and by sterile filtering, I get rid of the precipitants/fibers/aggregates as rkay447 mentioned.

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we donot sterilize buffers used for making cell lysates. Sometimes we may sterile filter the buffer.

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So why for cell lysis the washing PBS must be cold (4 degress)? what happens if I just wash with normal PBS stored at room temperature?

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Washing and extracting in 4C reduces protease activity compared to RT.

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If I add in protease inhibitor into the lysis buffer, it is also better to get cell lysates using cold lysis buffer?

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