4 replies to this topic

[AllenChiu]

Have you noticed that once you added SDS loading buffer directly into the well/dish of cells to be harvested,the mixture will become so extremely sticky that are very difficult to be transfered to EP tubes?
What is the reason in your opinion?
Why those things won't happen when you add NP40/TritonX100 lysis buffer onto cells?
How did you solve this problem?
Have you ever noticed that when you collected cells into a EP tube first by trypsin and centrifugation,then discarded supernatant and added SDS loading buffer to cells,then cells formed clumps?
How did you solve this problem?
Any advises is appreciated,thank you in advence!

[Inmost sun]

the stickyness of lysed cells depends on cell type, cell status and amount of lysis buffer (the less the more sticky); I think it results from polymers such as cytoskeleton proteins (actin, microtubuli) and also polynucleotides mainly DNA; the latter can be digested by DNase I; if you do not want to spent money for DNase or do not want to contaminate your probes with this enzyme, try heating the probes to lose viscosity; be quick during loading the probes

[AllenChiu]

Inmost sun, on 30 August 2010 - 05:41 AM, said:

the stickyness of lysed cells depends on cell type, cell status and amount of lysis buffer (the less the more sticky); I think it results from polymers such as cytoskeleton proteins (actin, microtubuli) and also polynucleotides mainly DNA; the latter can be digested by DNase I; if you do not want to spent money for DNase or do not want to contaminate your probes with this enzyme, try heating the probes to lose viscosity; be quick during loading the probes

thank you very much!

[laurequillo]

AllenChiu, on 30 August 2010 - 05:25 AM, said:

Have you noticed that once you added SDS loading buffer directly into the well/dish of cells to be harvested,the mixture will become so extremely sticky that are very difficult to be transfered to EP tubes?
What is the reason in your opinion?
Why those things won't happen when you add NP40/TritonX100 lysis buffer onto cells?
How did you solve this problem?
Have you ever noticed that when you collected cells into a EP tube first by trypsin and centrifugation,then discarded supernatant and added SDS loading buffer to cells,then cells formed clumps?
How did you solve this problem?
Any advises is appreciated,thank you in advence!

I boil the cells for Two minutes, then I sonicate them for 15-25 sec, and boil them for another 5 min before I load them in a gel

[AllenChiu]

laurequillo, on 30 August 2010 - 05:44 AM, said:

AllenChiu, on 30 August 2010 - 05:25 AM, said:

Have you noticed that once you added SDS loading buffer directly into the well/dish of cells to be harvested,the mixture will become so extremely sticky that are very difficult to be transfered to EP tubes?
What is the reason in your opinion?
Why those things won't happen when you add NP40/TritonX100 lysis buffer onto cells?
How did you solve this problem?
Have you ever noticed that when you collected cells into a EP tube first by trypsin and centrifugation,then discarded supernatant and added SDS loading buffer to cells,then cells formed clumps?
How did you solve this problem?
Any advises is appreciated,thank you in advence!

I boil the cells for Two minutes, then I sonicate them for 15-25 sec, and boil them for another 5 min before I load them in a gel

thanks！