Ablation of Recycling Endosomes
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From Ang et al. 2004, JCB 167: 531
Stock solutions
Human Tfn-HRP
- 0.5 mg/ml in H2O + 20% glycerol
Acid Wash
- 150 mM NaCl
- 20 mM citric acid
pH 5.0, adjust with NaOH
DAB solution
weigh freshly to make a 10 mg/ml in PBS solution -> Dilute 1:100 to make 0.1 mg/ml DAB in PBS working solution, PROTECT FROM LIGHT
Other
30% H2O2
1% BSA in PBS
Protocol
Tfn labeling
- Serum starve VSVG-transfected cells in SF-DMEM 1h at 40 ºC
- Tfn-HRP 0.01 mg/ml (1:50 from 0.5 mg/ml stock) 45 min 40 ºC in SF-DMEM
- If fluorescent transferrin to be added: 50 ug/ml Tfn-Alexa (1:100 from 5 mg/ml stock) 45 min 40 ºC in SF-DMEM together with Tfn-HRP
- Wash SF-DMEM
- Chase 20 min 40 ºC
Ablation on ice
- Wash 2x ice-cold PBS
- Wash 2x 5 min with acid wash to remove surface-bound Tfn-HRP
- Wash 2x ice-cold PBS
- Incubate cells for 1 hr on ice in the dark with rocking as follows:
- ABLATION: 0.1 mg/ml DAB + 0.025% H2O2 (8.4 µl 30% solution per 10 ml DAB in PBS)
- CONTROL: 0.1 mg/ml DAB, no H2O2
- Wash 2x 1% BSA in PBS
Chase
- Add warm HPMI + 100 ug/ml cycloheximide, chase at 32 ºC 1.5 h
- Wash PBS, FIX
- VSVG trafficks to PM in control cells, in ablated cells remains in Golgi/RE