I need to check protein expression in primary hepatocytes after some treatment through WB analysis. I am facing problem regarding amount of protein in cell homogenate. The protein concentration is coming very less (Approx. 1 microgram/mL). Can you kindly provide me a protcol in details regarding WB analysis with primary cells? I am here sharing the method which i followed-
- Collected approx 2 million cells in a 1.5 mL of centrifuge tube.
- Washed twice with PBS/DBBS.
- Added 100 micro liter of lysis buffer and kept on ice for 30 mins.
- Vortexed vigorously.
- Sonicated for 1 min and centrifuged at 12,000 g for 10 mins.
- supernatant has collected to make sample for WB.
Thanks in advance.
I not see any big flaw in your protocol, personally I not use the sonication step but not looks is the problem. Your number of cells are a bit low but is feasible. Potential problems: Your lysis buffer not have protease inhibitors and your proteins is degradated.
Other possibility: Your culture have too many dead cells, the solution in that case is improve the culture conditions or reduce the time in culture of primary cells.
Other solution, but the improvement is going to be minor, is reduce the volumen of lysis buffer, in order to concetrate more your protein.
Also could improve the protocol if you vortex vigorously each 5 minutes during the 30 minutes of ice incubation to try to extract the maximun amount of protein possible.
If properly lysed, 1mio of any cells should give you roughly ~ 100 µg, so you should get to around 1mg/ml in protein assay, not 1000 times less.
So the basics: 1: You have enough cells, e.g. you see a clear cell pellet before adding lysis buffer? 2. If lysis works, your pellet after lysis centrifugation should be much smaller than the cell pellet. 3. Protein detection: Most assays do not detect such low amounts as 1 µg/ml....(e.g. lowest for BCA is 5µg/ml) You're sure there is no calculation error and you indeed already have 1mg/ml?
Best
Christian
Apart from mentioned solution above. Did you check the viability of hepatocytes before lysis ! In my experience lower protein yield can also be dying hepatocytes that are loosely attached. the usual yield I get with 1 million viable cells is between 1-mg/ml
Also: be sure that the protein of interest is in the soluble part (supernatant). Sometimes the are in the insoluble part (pellet). You can dissolve the pellet in sample buffer, heat it and run some of it on gel, if you are in doubt.
Dear Miguel Gallardo ,
Dear @Aditya Dandekar,
Thank you very much for your kind attention.
The cell viability before seeding becomes 80-85% usually. After one night (more than 12 hrs) incubation I used to treat cells with drugs. Cell viability after one night incubation becomes 70-80% on an average.
Dear Christian Gabriel ,
Thank you very much.
Before adding the lysis buffer, the pellet is quite big and visible. What amount of lysis buffer (I mean optimum volume) should I add in add in to the pellet?
50 to 100 should be fine I'd say. Minimum I would use is 100 µl per 10 mio cells, maximum depends on concentration you want to achieve. What protein assay are you using btw?
Olaf J.F. Schreurs
Dear Sir,
Definitely I will try the method which you have mentioned.
Thanks a lot.
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