TCA-DOCProtein Precipitation Protocols Protein Precipitation Methods to concentrate or eliminate interferences before electrophoresis or protein determination
(In almost all the cases, proteins get irreversible denaturation and lost of biological activity)
TCA-DOC Normal TCA Acetone Precipitation Ethanol Precipitation TCA-DOC / Acetone Acidified Acetone/Methanol TCA-Ethanol Precipitation PAGE prep-Protein clean-up
Chloroform Methanol
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 –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 –20ºC and then 15min 4ºC; or longer time at
4ºC without the –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 normal blue sample buffer colour.)
Acetone Precipitation
To eliminate acetone soluble interferences and protein concentration
1) Add to 1 volume
of protein solution 4 volumes of cold acetone. Mix and keep 10min –70ºC and at least 90min –20ºC.
(Suggestion: leave ON if the protein concentration is very low).
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) Dry samples
under vaccum (speed-vac) or dry air to eliminate any acetone residue (smell
tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.
Ethanol Precipitation
Useful method to concentrate proteins and removal of Guanidine
Hydrochloride before PAGE-SDS
1) Add to 1 volume
of protein solution 9 volumes of cold Ethanol 100%. Mix and keep at least
60min.at –20ºC. (Suggestion: leave ON).
2) Spin 15min 4ºC
in microcentrifuge 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) Wash pellet
with 90% cold ethanol (keep at –20ºC). Vortex and repellet samples
5min at full speed.
4) Dry samples
under vaccum (speed vac) or dry air to eliminate any ethanol residue (smell
tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.
TCA-DOC/Acetone
Useful method to concentrate proteins and remove acetone and TCA
soluble interferences
1. To one volume
of protein solution add 2% Na deoxycholate (DOC) to 0.02% final (for 100
µl sample, add 1 µl 2% DOC).
2. Mix and keep
at room temperature for at least 15 min.
3. 100% trichloroacetic
acid (TCA) to get 10% final concentration (preparation
of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat 4ºC.Be
careful, use gloves!!!).
4. Mix and keep
at room temperature for at least 1 hour.
5. Spin at 4ºC
for 10 min, remove supernatant and retain the pellet. Dry tube by inversion
on tissue paper.
6. Add 200 µl
of ice cold acetone to TCA pellet.
7. Mix and keep
on ice for at least 15 min.
8. Spin at 4ºC
for 10 min in microcentrifuge at maximum speed.
9. Remove supernatant
as before (5), dry air pellet to eliminate any acetone residue (smell tubes).
For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.
10. (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.)
Acidified Acetone/Methanol
Useful method to remove acetone and methanol soluble interferences
like SDS before IEF
1) Prepare acidified
acetone: 120ml acetone + 10µl HCl (1mM final concentration).
2) Prepare precipitation
reagent: Mix equal volumes of acidified acetone and methanol and keep at
-20ºC.
3) To one volume
of protein solution add 4 volumes of cold precipitation reagent. Mix and
keep ON at -20ºC.
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).
5) Dry samples
under vaccum (speed-vac) or dry air to eliminate any acetone or methanol
residue (smell tubes).
TCA-Ethanol Precipitation
Useful method to concentrate proteins and removal of Guanidine
Hydrochloride before PAGE-SDS
1) Dilute 10-25µl
samples to 100µl with H2O
Add 100µl
of 20% trichloroacetic acid (TCA) and mix (preparation
of 100% TCA: 454ml H2O/kg TCA. Maintain in dark bottleat
4ºC.Be careful, use gloves!!!).
2) Leave in ice
for 20min. Spin at 4ºC for 15 min in microcentrifuge at maximum speed.
3) Carefully discharge
supernatant and retain the pellet: dry tube by inversion on tissue (pellet
may be difficult to see).
4) Wash pellet
with 100µl ice-cold ethanol, dry and resuspend in sample buffer.
5) In case there
are traces of GuHCl present, samples should be loaded immediately after
boiling for 7 min at 95°C
6) (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.)
PAGE prepTM Protein Clean-up and Enrichment Kit
- PIERCE
The PAGEprep™ Kit enables removal of many
chemicals that interfere with SDS-PAGE analysis: guanidine, ammonium sulfate,
other common salts, acids and bases, detergents, dyes, DNA, RNA, and lipids.
PIERCE: #26800
- PAGE prepTM Protein Clean-up and
Enrichment Kit (pdf)
Chloroform
Methanol Precipitation
Useful method for Removal of salt and
detergents
1) To sample of starting volume 100 ul
2) Add 400 ul methanol
3) Vortex well
4) Add 100 ul chloroform
5) Vortex
6) Add 300 ul H2O
7) Vortex
8) Spin 1 minute @ 14,0000 g
9) Remove top aqueous layer (protein is between layers)
10) Add 400 ul methanol
11) Vortex
12) Spin 2 minutes @ 14,000 g
13) Remove as much MeOH as possible without disturbing pellet
14) Speed-Vac to dryness
15) Bring up in 2X sample buffer for PAGE
Reference: Wessel, D. and Flugge,
U. I. Anal. Biochem. (1984) 138, 141-143
|
|
|
|
|
|
|
Copyright
©, 2002, The Hebrew University of Jerusalem. All Rights Reserved.