Fourty ug of lysed proteins are mixed with 5x loading dye (sample buffer) and boiled for 5 min. After cooling down the sample at RT and prior to loading, the sample appears as aggregates/cluster and a smear results following electrophoresis. The cultured cells (approximately 5 million) were prepared as follows: cells were trypsinized and the cell pellet was washed twice. Then, 100 ul triple-deterget lysis solution are added and the sample was incubated on ice for 1 hour with periodic vortexing. The sample was centrifuged and the supernatant was removed. Protein concentration was an average of 8 ug/ul. The sample looked fine at this point. It was not viscous or clustery. 4x sample buffer was added to a final concentration of 1x to 40 ug of protein sample, which was boiled for 5 minutes. The sample was cooled down at RT and, when loaded on bis-acrylamide gel, there were aggregates. Was the protein concentration high? Was the lysis buffer bad? How about the sample buffer?
To make sure, I get this right: You lyse the cells, add sample buffer, heat it and then load the gel?
What you most likely have there is genomic DNA which precipitates out of the solution and get very viscous. Do you have a sonicator available in your lab? Sonicating the sample for a few seconds usually breaks the DNA into small pieces, so loading and running the gel is not a problem.
To make sure, I get this right: You lyse the cells, add sample buffer, heat it and then load the gel?
What you most likely have there is genomic DNA which precipitates out of the solution and get very viscous. Do you have a sonicator available in your lab? Sonicating the sample for a few seconds usually breaks the DNA into small pieces, so loading and running the gel is not a problem.
Also, try running it with a fresh loading buffer.
If I am reading your protocol right, your protein may also be forming aggregates during the boiling step that are not soluble in SDS. You try running the gel without boiling first.
In my hands, viscous samples are related with precipitated DNA and/or poor cell lysis. I would add to previous suggestions to drag the tube with your protein sample + loading buffer across the surface of the rack, vigorously and repeteadly, to ensure full break of aggregates and DNA. Boiling sample seems not to be mandatory, so I would compare samples with and without boiling. And finally, double check your buffers, just in case there's any reagent susceptible to generate aggregates.
Good luck!
Just out of curiosity: what is in your gel loading buffer? Do you have any other denaturing agents aside from SDS, such as beta-Mercaptoethanol? I do not think you are adding too much proteins to your loading buffer, but I would suggest putting less amount of protein and boil it to see if there is too much protein in the mixture. Also, your lysis buffer may not be compatible with your loading dye so you may want to use different lysis buffer.
Thank you all. The DNA is sheared by a syringe repeatedly 20 times until viscosity disappears. The samples is then centrifuged to remove any clusters. I will try no boil and prepare fresh buffers. The loading buffer is a standard one with SDS and b-ME.
Check also presence of potassium, magnesium and other ions in the mix that produce non-soluble salts with SDS. Good luck!
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