Protein Precipitation Protocols

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 –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 at least 20min –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 10min.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℃

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 100μl

2)Add 400μl methanol

3)Vortex well

4)Add 100μl chloroform

5)Vortex

6)Add 300μl H₂O

7)Vortex

8)Spin 1 minute @ 14,0000 g

9)Remove top aqueous layer (protein is between layers)

10)Add 400μl 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