Shall I change to any other harsh lysis buffer? Also I have run the gel on SDS PAGE I am getting one single band between 50- 75 Kda. Is it because of improper denaturation? Please help.
First you have to start with enough cells - min 100'000 cells and use protease inhibitor, such as PMSF. Don't dissolve in too much buffer - about 100 ul is maximum. Load you gel with minimum 20 ul. If your Coomassie is old - prepare new stock for better visualisation.
Can I ask you from which kind of flask/plate you start and the lysis volume you incubate the cells in?
In our experience, even if we use a NP-40 based buffer, a volume of 70-150 ul works well with 25cm2/75cm2 flasks. In this way you should obtain protein concentrations ranging around 10 ug/5ul, so easy to load 30ug for a good western.
Please consider that MSCs are larger than standard cultured cells (e.g. Hela) so even a confluent flask has a lower yield (in numbers) than expected.
Protease inhibitors may be in a cocktail (many are readily available as 100x solutions from different providers), and not limited to PMSF alone, as tipically MSCs and culture media bear also metaloproteinases and other proteolytic activities which are insensitive to PMSF.
Hey,
how dou you prepare your lysates? The prominent band at 55-70 kDa can be eighter BSA (in case you have not properly washed your samples), further more if it is visible in slots without lysate it could be keratin from your skin.
Ripa buffer normally yields good protein amounts. so normally you should get good protein amounts out of a T75.
Hi, Thanks for the reply. I am using 1 million cells and dissolve it in Approximately 150ul of RIPA lysis buffer ,sometimes less also. and I use a protease inhibitor cocktail available from Sigma. Still the concentration remain very poor. Also I have used different type of loading dyes , thinking of improper denaturation but still I am getting one single band between 50-75 kda. I am guessing its albumin. Can anybody suggest me what could be the reason that i am getting only single band after transfer. Thanks.
hi,
make sure you work on ice for all steps. Make sure your buffer if not very old and inhibitors are added just prior to use.
I tend wash my cells first a couple of times with cold 1xPBS to get rid off any medium proteins.
I tend to trypsinise my cells and pellet them in an eppendorf tube prior lysis to ensure i can have my samples as concentrated as possible.
RIPA should extract the majority of proteins. The fact you are using a cocktail of protease inhibitors does help.I tend to sonicate the samples to get rib off any nucleic acid and spin down to pellet the debri. Then transfer the supernatant into a new tube.
After measuring the protein concentration I aliquot the required amount into a tube and add protein loading buffer.
depending on what you are using it for 20-30µg/lane is good for an SDS page. However, it may also depend on the abundance of the protein you are after.
I denature my samples in reducing protein buffer containing beta-mercaptoethanol and denature the proteins by heating at 98C for 2min. then put back on ice to cool down, spin down briefly to collect sample prior loading.
I hope that helps
good luck
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