Jul 07, 2022

Public workspaceTX-100 FRACTIONATION PROTOCOL

DOI
dx.doi.org/10.17504/protocols.io.5qpvob21zl4o/v1
  • Scott Vermilyea1
  • 1University of Minnesota
Jane Balster
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DOI: dx.doi.org/10.17504/protocols.io.5qpvob21zl4o/v1
Protocol Citation: Scott Vermilyea 2022. TX-100 FRACTIONATION PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvob21zl4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 03, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 63788
Keywords: insoluble proteins, soluble proteins, TX-100 FRACTIONATION, brain, ASAPCRN
Abstract
To isolate insoluble and soluble proteins from dissected brain regions frozen for biochemical analysis.
Attachments
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452-952.docx
19KB
Materials
Materials

  • phosphatase
  • protease inhibitors
AB
1x TNE (TNE) + Inhibitors1 mL
2x TNE500 L
100x HALT inhibitor10 µL
100x HALT EDTA10 µL
H2O to final480 µL
AB
Complete 2x TNE (2x CTNE) + Inhibitors1 mL
2x TNE500 µL
1% SDS100 µL
0.5% NP-40100 µL
0.5% DOC100 µL
100x HALT inhibitor10 µL
100x HALT EDTA10 µL
H2O to final180 µL
Thaw Tissue
Thaw Tissue
8h
8h
If tissue stored at Temperature-80 °C , place in Temperature-20 °C for at least Duration04:00:00 or DurationOvernight to thaw prior to homogenization.

8h
Overnight
Prepare 1X TNE
Prepare 1X TNE
Prepare 1X TNE with phosphatase and protease inhibitors.
Homogenize Tissue
Homogenize Tissue
Weigh tissue out in mg.
Add in 10 volumes of 1X TNE.
  1. Amount10 µL of TNE per Amount1 mg of tissue.
  2. Ex: Amount50 mg tissue = Amount500 µL of 1X TNE.
Either by mechanical (Dounce) or homogenizer machine, homogenize tissue gently and TemperatureOn ice .
This is TNE crude lysate (no detergents).
Prepare for Soluble v Insoluble
Prepare for Soluble v Insoluble
3h 20m 36s
3h 20m 36s
Take specific volume of tissue in TNE and add in equal volume of 1X TNE w/ 2% Triton X-100 (Tx100).
  • Ex. Amount150 µL of TNE tissue + Amount150 µL 1X TNE+2% Tx100.

Sonicate @ Temperature4 °C .
3 pulses:Duration00:00:10 ON / Duration00:00:02 OFF.

12s
Spin down.
Centrifigation
Option 1: Centrifigation16000 x g, 4°C, 00:15:00 .

15m
Option 2: Centrifigation20000 x g, 4°C, 01:00:00 .

1h
After spin:
Supernatant = soluble. Save supernatant and add equal volume of complete TNE (cTNE).

  1. Sonicate @ Temperature4 °C : 3 pluses: Duration00:00:10 ON / Duration00:00:02 OFF.
  2. Boil: Duration00:10:00 at Temperature95 °C .
  3. Spin down: Centrifigation16000 x g, 4°C, 00:15:00 .
  4. Supernatant from this is the soluble fraction.
25m 12s
Pellet = insoluble.
Wash pellet in Amount150 µL of 1X TNE+1% Tx100.
Note
Note: Same volume that was used above from TNE.


Wash
Resuspend pellet via pipette.
Pipetting
Spin down.

  1. Option 1: Centrifigation16000 x g, 4°C, 00:15:00 .
  2. Option 2: Centrifigation20000 x g, 4°C, 01:00:00 (Centrifigation25000 x g ) .

1h 15m
Centrifigation
Resuspend pellet in Amount75 µL to Amount100 µL of cTNE.

Sonicate @ Temperature4 °C : 3 pluses: Duration00:00:10 ON / Duration00:00:02 OFF.

12s
Boil: Duration00:10:00 at Temperature95 °C .

10m
Spin down: Centrifigation16000 x g, 4°C, 00:15:00 .

15m
Centrifigation
Supernatant from this is the insoluble fraction.