Bitan:Biotinylation of antibodies)

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Biotinylation of mAb211

-          mAb211 stock = [200ug/mL) in PBS

-          Biotin Labeling Reagent Used: NHS-PEG12-Biotin (from ThermoScientific)

-          Amount of Biotin Reagent Used for 40mmol excess BR (labeled as 2X):

o   0.030mL x 0.2mg/mL x 1mmol IgG/150,000 mg IgG x 40mmol BR/mmol mAb211 = 0.0000016mmol BR

o   0.0000016mmol BR x 1,000,000uL/L x L/250mmol = 0.0064uL BR from original stock\

-          Amount of Biotin Reagent Used for 60mmol excess BR (labeled as 3X):

o   0.030mL x 0.2mg/mL x 1mmol IgG/150,000 mg IgG x 60mmol BR/mmol mAb211 = 0.0000024mmol BR

o   0.0000024mmol BR x 1,000,000uL/L x L/250mmol = 0.0096uL BR from original stock

-          Take 1uL from original stock vial (use syringe from Aida’s draw under column)

-          Add 999uL DMSO to make 1000X dilution

o   Always use dry solvent for BR since NHS-ester reactive group is susceptible to hydrolysis with exposure to moisture.

-          6.4 uL of 1000X diluted BR added to 30uL of mAb211 (2X)

-          9.6uL of 1000X diluted BR added to 30uL of mAb211 (3X)

-          Both eppendorfs were incubated with BR on ice for 2 hours

-          Unreacted biotin removed by running solution through micro-column

o   To prepare micro-column remove bottom seal and loosen cap

o   Centrifuge for 1.5 min at 14000 rpm

o   Solution discarded and slanted portion marked

o   Sample loaded on column

§  2X – approx 36uL

§  3X – approx 40uL

o   Sample allowed to absorb on column

o   15uL of PBS used as stacking buffer to fully elute protein since <70uL sample loaded on column.

-          Final volumes:

o   2X – approx 52uL (30 + 6.4BR + 15PBS)

o   3X – approx 55uL (30 + 9.6BR + 15PBS)

 

Biotinylation Check

-          Prepare membrane (Nitrocellulose membrane)

o   Do not touch membrane

o   Cut to appropriate size

o   Mark container

-          Prepare dilutions of protein sample

o   For 2x and 3X prepare a 10X and 100X dilution

§  10X: 1uL of original diluted in 9 uL PBS

§  100X: 1uL of 10X diluted in 9uL PBS

-          Prepare blocking reagent. Use advance blocking reagent (1gram in 50mL milliQ)

o   Store at 4 deg C till use

-          Blot Protein on membrane (2uL)

o   Triplicates for undiluted, 10X dilution, and 100X dilution

 

-          Membrane dried to prevent spread of protein across membrane.

o   Best to keep covered in hood to dry. No need to refrigerate. Prevent accumulation of moisture

-          Block with Advance Blocking Reagent ( 2% - 1g in 50mL) for 90 min

o   Incubate at room temperature on rotator

o   Make sure membrane is moving

o   Used 10mL

-          Add 1uL of streptavidin HRP to blocking solution and incubate for 60-75min

o   1:10000 dilution for streptavidin HRP (1uL in 10mL)

o   Dilute in blocking buffer

-          Wash thoroughly: complete 4, 5 min washes

o   Wash 4 times with 10mL PBST for 5 minutes each

§  PBST: 50mL PBS + 25uL Tween20

-          Prepare ECL substrate (reacts with streptavidin-HRP/biotin complex to produce chemiluminence which is then detected by the imager.)

o   Take kit out of fridge for at least 10 min

o   Mix 200uL of each substrate and incubate 5 min with membrane

§  Roll solution to spread over entire membrane

o   Keep ECL substrate wrapped in foil and cover the membrane containing box with foil as well. ELC substrate is highly sensitive to light and will degrade quickly if exposed to light

-          Image in the Spencer Lab imager

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