娌夋穩铔嬬櫧璐ㄧ殑甯哥敤鏂规硶锛?/p>
TCA
銆佷箼閱囥�佷笝閰矇娣�铔嬬櫧鎿嶄綔姝ラ锛?/p>
TCA-DOC
For precipitation of very low protein concentration
1) To one volume of protein solution, add 1/100 vol. of 2% DOC (Na deoxycholate,
detergent).
2) Vortex and let sit for 30min at 4oC.
3) Add 1/10 of Trichloroacetic acid (TCA) 100% vortex and let sit ON at 4oC
(preparation of 100% TCA: 454ml H2O/kg TCA. Maintain
in dark bottleat 4oC.Be
careful, use gloves).
4) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge
supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be
difficult to see).
[OPTION: Wash pellet twice with one volume of cold acetone
(acetone keep at
鈥?/p>
20oC). Vortex and repellet samples 5min at full speed between
washes].
5) Dry samples under vaccum (speed vac) or dry air. For PAGE-SDS, resuspend
samples in a minimal volume of sample buffer. (The presence of some TCA can give
a yellow colour as a consequence of the acidification of the sample buffer ; titrate
with 1N NaOH or 1M TrisHCl pH8.5 to obtain the normal blue sample buffer colour.)
Normal TCA
To eliminate TCA soluble interferences and protein concentration
1) To a sample of protein solution add Trichloroacetic acid (TCA) 100% to get 13%
final concentration. Mix and keep 5min
鈥?/p>
20oC and then 15min 4oC; or longer time at
4oC without the
鈥?/p>
20oC step for lower protein concentration. Suggestion: leave ON if
the protein concentration is very low.
(preparation of 100% TCA: 454ml H2O/kg TCA. Maintain
in dark bottleat 4oC.Be
careful, use gloves).
2) Spin 15min 4oC in microfuge at maximum speed (15000g). Carefully discharge
supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be
difficult to see).
3) For PAGE-SDS, resuspend samples in a minimal volume of sample buffer. (The
presence of some TCA can give a yellow colour as a consequence of the acidification
of the sample buffer ; titrate with 1N NaOH or 1M TrisHCl pH8.5 to obtain the