Lyse cells with TCA - (Feb/11/2007 )

Dear all,

To do a HPLC measurement in tissue, our lab uses TCA as a homogenizing buffer. We wanna do the measurement in vitro (cell lines), does anyone know if we can use TCA to LYSE the cells? if not, in addition to TCA, what else do we need in order to facilitate the cell lysing? (say like syringe? sonication? ...etc), if we wanna stick onto the TCA protocol. Thx Thx Thx.

As I know TCA used for precipitation of proteins but is it work good for homogenise cells. It is very surprising for me. And is it really good for HPLC analysis? Whats about coprecipitated lipids and other non protein substances? They may clogged your HPLC column really!

Here protocol? which we use for mammalian cell lysis for western blot

Protein extraction from mammalian cells Wash cells twice with PBS and aspirate the PBS.

2. Add 1 ml of PBS to each plate, scrape the adherent cells with a rubber policeman to dislodge the cells into the PBS, and collect the cell suspension in a microcentrifuge tube.

3. Centrifuge cells at approximately 8,000 rpm in a microcentrifuge for 10 min.

4. Aspirate the supernatant and estimate the cell pellet volume*.

5. Add 1 μl of 10X Lysis Buffer plus Protease Inhibitors per 10 μl of cell pellet volume (or add an equal volume of 1X Lysis Buffer with Protease Inhibitors).

6. Vortex for a few seconds to mix well.

7. Add 0.2 volumes of 1X Lysis Buffer/DNase I Solution.

8. Incubate the cell suspension at 37°C for 10 min.

9. After the incubation, add 0.2 volumes of 5X Protein Loading Sample Buffer.

10. Heat the samples in a boiling water bath for 1 to 2 min.

12. Use between 1 μl to 20 μl of samples for Western analysis.


Solutions
Aprotinin Stock (200X)
10 mg/ml Aprotinin
Store at -20°C

Leupeptin Stock (100X)
1 mg/ml leupeptin
Store at -20°C

Pepstatin Stock (400X)
Prepare in 100% Ethanol
3 mg/ml Pepstatin
Store at -20°C

PMSF Stock (500X)
Prepare in 100% Ethanol
43 mg/ml PMSF
Store at -20°C

Lysis Buffer (10X)
30 mM MgCl2
100 mM NaCl
5% (v/v) IGEPAL CA-630 (Sigma, replaces NP-40)
100 mM Tris-HCl, pH 8.0
Store at 4°C

PBS
pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
* - Typically, the cell pellet volume is between 10-50 μl.

I think that pulse sonication also really help with lysis buffer application

As summary and fast lysis try to add detergent, DNAse, ( in conc mention above) and pulse sonication for prepare for western or SDS-PAGE
Good Luck