Version 1.00, Jan 15, 2015
Materials
Buffers:
Buffer A: 150 mM NaCl, 100mM Na2HPO4, pH 7.4
Buffer C: 150 mM NaCl, 100mM Na2HPO4, pH 6.0
(Titrate with NaOH or HCl to pH)
Reagents, Columns, and Filters:
Vendor
Product Name
Catalog Number
Solulink
10x Turbolink Catalyst Buffer
S-2006-105
Solulink
Sulfo S-4FB Crosslinker
S-1008-105
Solulink
S-HyNic Crosslinker
S-1002-105
Sigma
MES sodium salt
M3058-25G
Thermo Scientific Zeba Spin Desalting Columns
87768 and 87766
Amicon
Ultra-0.5mL Centrifugal Filters
UFC510024
Vivaproducts
Vivaspin 500 Centrifugal Concentrator VS0111
Sigma
2-Hydrazinopyridine dihydrochloride
H17104-10G
Procedure:
Stage 1: Modification of Amino-Oligonucleotide with S-4FB
A.
Preparing Amine DNA for modification (EtOH precipitation and resuspension)
a) Order DNA from IDT-DNA with 5’ Amine modification. HPLC purification is not
necessary; make sure to order at least 100 nmole scale synthesis.
b) Dissolve DNA in 100 uL of water.
c) Extract DNA with equivalent volume (100 uL) of chloroform. Add chloroform to DNA
and vortex to mix. Spin down with centrifuge and move aqueous fraction to another tube.
d) Repeat step C two times.
e) Add 1/10th volume (10 uL) of 3 M NaCl to extracted aqueous fraction. Then add 2.5
(250 uL) volumes of ice cold 100% ethanol.
f) Incubate solution at -20 C for 30 min - 1 hour.
g) Spin in refrigerated centrifuge @ 4C for 30 min at max speed.
h) Remove ethanol from pellet on ice. Wash once with ice cold 70% EtOH. Remove as
much liquid after wash as possible
i) Dry pellet w/ speedvac, or on bench with cap uncovered. Resuspend with Buffer A at ~
25 ug/uL (estimate volume with original lyophilized DNA weight).
j) Measure DNA concentration with Nanodrop. Dilute DNA 1:100 before measurement to
get into dynamic range of Nanodrop. Measure A260, and use extinction coefficient to
calculate DNA concentration, and total amount in nanomoles.
B.
Reaction of DNA with Sulfo-S4FB
a) Resupend 1 mg Sulfo-S4FB in 40 uL of anhydrous dimethylformamide or DMSO.
b) Using the total amount of nanomoles of DNA obtained in step K before, the amount of
Sulfo-S4FB needed in uL is calculated as:
i) (nmol of DNA)*349*40*(Molar Ratio of S4FB to DNA)/10^6
(1) 349 is molecular weight of Sulfo-S4FB
(2) Molar concentration of Sulfo-S4FB in nmol/uL : 10^6/349/40
(3) Typically use 15 for molar ratio
c) React DNA with Sulfo-S4FB for at least 2 hours in Buffer A.
d) Purify DNA with Vivaspin 500 centrifugal filter. Note: make sure molecular weight
cutoff is small enough for DNA (e.g. use 5 kda for 20mer or greater)
i) Add buffer C to the DNA/S4FB conjugation reaction until total volume is 500 uL,
add to the centrifugal filter, and spin at 13000 g for 10 minutes.
ii) Discard flowthrough and add buffer C to centrifugal filter until volume ~500 uL. Spin
at 13000 g for 10 min.
iii) Repeat step (ii) 3 additional times.
e) Pipette out concentrate after spins, and bring total volume up to 50 uL with Buffer C.
C.
Quantifying molar substitution ratio of oligo with 2-HP
a) Prepare a 0.5 mM working solution of 2-hydrazinopyridine·2HCl (2-HP) solution in 0.1
M MES buffer, pH 5.0 as follows:
i) Weigh 5 mg of 2-hydrazinopyridine·2HCl.
ii) Create a 50 mg/mL solution of 2-hydrazinopyridine·2HCl (M.W. 182.1) in 100 µl
nuclease-free H2O.
iii) Add 91 µl of this solution to a 50 ml conical tube containing 50 ml of 100 mM MES
Buffer, pH 5.0 to make 0.5 mM 2-HP working solution
iv) Protect the solution from light and keep refrigerated. This solution remains stable for
up to 30 days at 4° C.
b) Prepare a 2-HP blank solution by adding exactly 2 µL water to exactly 18 µL 2-HP
Working solution
c) Prepare a 4FB-oligo MSR sample by adding exactly 2 µL 4FB-modified oligo to exactly
18 µL 2-HP working solution in a clean reaction tube; label ‘4FB-Oligo’.
d) Vortex solutions from b and c. Incubate the 2-HP blank and 4FB-Oligo reactions at 37 C
for 60 minutes.
e) After the incubation period, centrifuge both tubes at 15,000 x g for 15 seconds to collect
condensation at the bottom of the tube. Gently vortex to thoroughly mix.
f) Use UV-VIS module on nanodrop. Blank with 2-HP blank solution, and read the 1mm
pathlength A360 and A260 of 4FB-oligo reaction. If A260 reading is much higher than 1,
make sure to use the 0.1 mm path length to get a more accurate reading.
D.
Quantify molar substitution ratio (MSR)
MSR can be calculated as:
A360/A260*(DNA extinction coefficient/24500)
uL of DNA per 100 ug can be calculated as:
5*(100ug)/(150000 Da)*1000/(MSR*[Concentration of DNA after S4FB DNA in
mM])
Can pause here, S4FB modified DNA can be stored at 4 degrees for 1 year.
E.
Stage 2: Modification of Antibody with S-HyNic
A.
Antibody preparation and buffer exchange antibody
a) Secondary antibodies were obtained from Jackson Immunoresearch.
b) Buffer exchange secondary antibodies into buffer A with 40 kDA Thermo Scientific Zeba
column.
i) Best to do use 2 mL column for this (Pierce: 87768) this can exchange ~ 900 ul of
antibody.
ii) Bring antibody to ~ 1 mg/mL (check with nanodrop protein A280). Antibody can be
stored at 4 degrees after buffer exchange for at least 1 month.
B.
a)
C.
a)
b)
c)
d)
Dissolve S-HyNic reagent
Dissolve S-HyNic reagent (1mg) in DMF by adding 350uL DMF to 1 mg S-HyNic and
pipetting up the S-HyNic solution up and down for 60 seconds. This solution is good for
~1-2 weeks if stored under anhydrous conditions.
Modify IgG with S-HyNic Reagent and Buffer Exchange
Add 2.6 uL of the S-HyNic reagent solution to 130 uL secondary antibody (buffer
exchanged in step B) in Buffer A.
Incubate the S-HyNic/Antibody solution at room temperature for 2 hours.
10 minutes before the reaction completes, prepare spin columns (Thermo Scientific,
#89882)
i) Remove the column’s bottom closure and loosen cap (do not remove cap)
ii) Place the column into a microcentrifuge collection tube and centrifuge at 1500g for 1
min to remove the storage solution.
iii) Discard flow-through and replace the column back into the collection tube.
iv) Place a mark on the side of the column where the compacted resin is slanted upward.
v) Add 300 uL of Buffer C on top of the resin. Place the column in the centrifuge with
the mark facing outward. Centrifuge at 1500g for 1min and discard flow-through.
Repeat this step two additional times.
vi) Place the column in a new microcentrifuge collection tube, remove the cap.
Apply the reaction solution to the top of the compact resin bed.
e) Place the column in the centrifuge with the mark facing outward and centrifuge at 1500g
for 2 min to collect the sample. Discard the column after use, save the flow-through.
Stage 3: Formation of Conjugate and Purification
A.
Conjugate Formation
a) Add 1.5 times of the volume calculated in Stage 1-D to the recovered antibody solution.
i) For example, if 5 uL of 4FB-DNA needs to be added to 100 uL of secondary antibody
solution, add 7.5 uL of 4FB-DNA to the recovered secondary antibody solution.
b) Add 1/9 of the total volume of antibody/DNA of Turbolink Catalyst Buffer (10x) to the
antibody/DNA solution.
c) Incubate the solution at room temperature for 2 hours.
B.
Conjugate Purification
a) Place a filter in a collection microcentrifuge tube and apply the reaction solution to the
filter. Add 1x PBS to the filter to make the total volume in the filter 500 uL.
i) For full size antibodies (~150 kda mw) use Amicon Ultra 0.5 mL (UFC 510024)
ii) For Fab 2’ fragments and scFVs use Vivaspin 500 (VS0101)
b) Centrifuge the filter for 5 minutes at 14000 g.
c) Discard flow-through and add 1x PBS to the filter to make the total volume of the liquid
in the filter 500 uL. Centrifuge the filter at the same speed of the above step for 5 min.
Repeat this step twice.
d) Pipette out concentrate and measure out concentrated volume (should be <100 uL), bring
up total volume to 100 uL with 1x PBS.
i) It helps to wash the filter walls with the excess 1x PBS used to bring the total volume
up to 100 uL, in case there was some liquid which was not removed by pipetting.
ii) Measure the protein/DNA concentration using Nanodrop.
(1) Measure the protein/DNA bond using A360, the extinction coefficient of this
bond is ~ 23000. The 1 mm absorbance should be on the order of 0.1.
3. Reference
Antibody-Oligonucleotide All-In-One Conjugation Kit Technical Manual
http://www.solulink.com/products/ptm/A-9202-001AntibodyOligonucleotideAllInOneConjugationKit.pdf
Zeba Spin Desalting Columns and Plates, 7K MWCO
https://www.piercenet.com/instructions/2161515.pdf
Amicon Ultra-0.5 mL Centrifugal Filters for DNA and Protein Purification and Concentration
http://www.emdmillipore.com/US/en/product/Amicon-Ultra-0.5%C2%A0mL-CentrifugalFilters-for-DNA-and-Protein-Purification-and-Concentration,MM_NFC82301?isCountryEMD=yes#overview
Vivaspin 500 uL and 2mL
http://www.vivaproducts.com/downloads/vivaspin-2-500-operating-instructions.pdf