General Intracellular Nuclear Target Flow Protocol

Protocol for Nuclear Targets (Nuclear Envelope and Nuclear Matrix)

  1. Optional: Perform cell surface staining as described in the previous section and/or the cytoplasmic target staining described above.
  2. Fix the cells by adding 100 µL fixation solution (such as 4% PFA) to each sample for 10-15 minutes.   
  3. Permeabilize cells by adding 100 µL of a permeabiliation buffer to every 1 x 106 cells present in the sample.  Mix well and incubate at room temperature for 15 minutes.
    1. For cytoplasmic targets, use a gentle permeabilization solution such as 1X PBS + 0.5% Saponin or 0.5% Tween-20.
    2. To maintain the permeabilized state throughout your experiment, use staining buffer + 0.1% of the permeabilization reagent (i.e. 0.1% Tween 20 or 0.1% Saponin).
  4. Following the 15 minute incubation, add 2 mL of the staining buffer + 0.1% permeabilizer solution to each sample.
  5. Centrifuge for 5 minutes at 400 RCF.
  6. Discard supernatant and re-suspend in 1 mL of staining buffer + 0.1% permeabilizer.
  7. Stain each sample at 1 µL/ 1 x 106 cells of primary antibody or 1-3 µL/ 1 x 106 cells for directly conjugated antibodies. Mix well and incubate on ice for 30 minutes- 1 hour. Gently mix samples every 10-15 minutes.
  8. Following the primary/conjugate incubation, add 2 mL/sample of staining buffer +0.1% permeabilizer and centrifuge for 5 minutes at 400 RCF.
  9. Remove supernatant and re-suspend each sample in 2 mL staining buffer +0.1% permeabilizer, repeat wash for 5 minutes at 400 RCF.

If using a directly conjugated antibody, after the second wash, re-suspend cell pellet to a final volume of 500 µL per sample and proceed with flow analysis.

General Solution Recipes

1X Running buffer

 

1X Transfer buffer

 

4% Paraformaldehyde (PFA)

12.5 mM Tris-base

1.51 g

 

25 mM Tris-base

3.03 g

 

Paraformaldehyde

4 g

100 mM Glycine

7.5 g

 

192 mM Glycine

14.4 g

 

1X PBS

To 100 mL

0.05% SDS

0.5 g

 

20% Methanol

200 mL

 

Gently heat in a fume hood while stirring. Do not boil. Cool to room temperature before use

Water

To 1000 mL

 

Water

To 1000 mL

 

 

 

 

 

 

1X TBST

 

1X PBS

 

1X Laemmli Sample Buffer

20 mM Tris-base

2.4 g

 

10 mM Na2HPO4

2.17 g

 

62.5 mM Tris-HCl pH 6.8

150 mM NaCl

8.7 g

 

1.8 mM KH2PO4

0.24 g

 

10% Glycerol

0.1% Tween-20

1 mL

 

140 mM NaCl

8 g

 

2% SDS

Water

To 1000 mL

 

2.7 mM KCl

0.2 g

 

0.005% Bromophenol blue

 

Adjust pH to 7.5

 

Water

To 1000 mL

 

 

 

 

 

 

Adjust to pH 7.4

 

Add reducing agent to 2.5% as required

 

 

 

 

 

 

0.1% Ponceau S Protein Stain

 

Western Blot Block Solutions

 

ICC Permeablization Buffer

Ponceau S

0.1 g

 

Non-fat Milk or BSA

1 or 5 g

 

Triton X-100

0.5 mL

Acetic Acid

1 mL

 

1X TBST

To 100 mL

 

PBS

99.5 mL

Water

To 100 mL