Cell surface biotinylation on Lysine residues

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  • EZ-Link sulfo-NHS-SS Biotin (Pierce): cleavable, will bind to all accessible Lysine residues; dissolve in H buffer to 1 mg/ml.
  • H buffer
  • PBS + 1 mM MgCl2 + 0.1 mM CaCl2 with 1% BSA [referred to as PBS(+)-BSA].
  • RIPA lysis buffer (150 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, pH 8.0. With proteinase inhibitors: 10 µg/ml of leupeptine, 10 µg/ml pepstatin, 1 mM PMSF).
  • 5X Laemmli Sample Buffer (diluted from 10x).
  • (optional) MESNA solution for stripping the bound biotin.
H buffer composition, pH 7.6 (It need around 310 ul NaOH 10N to reach Ph 7.6)
final volume (ml): 500
desired [] (mM) MW (g/mol) needed weight (mg)
NaCl 154 58.44 4499.88
Hepes 10 238.3 1191.50
KCl 3 74.56 111.84
MgCl2 1 203.3 101.65
CaCl2 0.2 110.99  (147.02) 5.55 (14.7)
glucose 10 180.16 900.80

 

MESNA solution composition, pH 8.6
final volume (ml): 100
desired [] (mM) MW (g/mol) needed weight (mg)
NaCl 100 58.44 584.4
TrisHCl (maybe means Tris) 50 157.6 (121.14) 788 (605.7)
MgCl2 1 203.3 20.33
CaCl2 0.1 110.99 (147.02) 1.11 (1.47)
MESNA 50 164.2 821

 

10X sample buffer Laemilli (LSB):

12.5% SDS

500 mM DTT (dithiothreitol)

300 mM Tris pH6.8

40mM EDTA

1.75% Glycerol

0.035% BPB (color blue)

Procedure

Seed CFBE-iCFTR WT-3HA cells 100.000/well on filters and keep them >5 days confluent with 250 ng/mL doxycycline on 12 well filter plates.

  • Wash the cells 3 times with ice-cold H buffer.
  • Incubate 15 min with (for 6 cm plate = 0.5 to 1 ml) (1 mg/ml) sulfo-NHS-SS Biotin in H buffer at 0°C. Optional: remove it with suction and repeat it.
  • Rinse twice with ice-cold PBS(+)-1% BSA, and incubate 10 min in PBS(+)- 1% BSA.
  • Optional: perform study of endocytosis.
  • Optional: treat cells with MESNA 3 times 20 min on ice to remove biotin from the cell surface
  • Lyse the cells with(0.5 to 1 ml) RIPA buffer containing protease inhibitor on ice (less than 10 min)
    • Cut out the 4 filters and put into 400 uL RIPA in a pre-cold eppendorf for 5-10 min
    • Vortex throughfully, pipet up and down 5 x and remove the filters.
  • Vortex and centrifuge at 4°C for 10 min at 12000 g (supernatants can be frozen and kept at -80°C).
  • Incubate the supernatant with 20 µl BcMag monomeric avidin magnetic beads (#MMI-102) (wash the beads 3 times with RIPA + protease inh before use) and rotate for 1 h at 4°C.
  • Use the magnetic separator and wash beads by resuspending in 1 ml of lysis buffer. Repeat twice more.
  • Elute the protein with 50 µl + 20 of 5X Laemmli Sample Buffer (diluted from 10x LSB) + 6mM biotin (B4505-1G) and incubate for 10-20 min at RT. Vortex it.
  • Centrifuge the samples at 14000 RPM, RT for 2 min, save the supernatants and perform a 4%-7% SDS-PAGE (when revealing CFTR or other membrane protein, use also an antibody against a cytosolic protein – Hsc70 or other – to check that NHS-SS Biotin has not entered the cells).

 

ImageLab 6.0

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ImageLab 6.0

https://app.box.com/s/z4grf20x1pd4u5df11n8

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http://www.nature.com/articles/srep11386

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http://smart.embl-heidelberg.de/smart/domain_table.cgi

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http://93.174.95.27/

http://libgen.io/

http://gen.lib.rus.ec/

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