G BIOSCIENCES Amine reactive biotin reagents, HOOK Owner's Manual

132PR‐03
G-Biosciences ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected]
A Geno Technology, Inc. (USA) brand name
HOOK™ Sulfo‐NHS‐LC‐Biotin
For the coupling of biotin to protein amine groups
(Cat. # BS‐07)
think proteins! think G-Biosciences
www.GBiosciences.com
INTRODUCTION ................................................................................................................. 3
ITEMS SUPPLIED ................................................................................................................ 4
STORAGE CONDITION ........................................................................................................ 4
SPECIFICATIONS ................................................................................................................. 4
PRECAUTIONS .................................................................................................................... 4
ADDITIONAL ITEM(S) REQUIRED........................................................................................ 4
PREPARATION BEFORE USE ............................................................................................... 4
A. PROTEIN SAMPLE PREPARATION................................................................................... 5
B. CALCULATION OF QUANTITY OF BIOTIN AGENT NEEDED FOR CONJUGATION ............. 5
C. PREPARATION OF BIOTIN AGENTS ................................................................................ 5
D. BIOTIN CONJUGATION REACTION ................................................................................. 6
E. REMOVAL OF UNCONJUGATED BIOTIN AGENTS ........................................................... 6
F. ESTIMATION OF BIOTIN INCORPORATION EFFICIENCY .................................................. 6
IMPORTANT INFORMATION .......................................................................................... 6
PROTOCOL 1: CUVETTE PROTOCOL ............................................................................... 7
PROTOCOL 2: MICROPLATE PROTOCOL......................................................................... 7
CALCULATIONS .............................................................................................................. 8
TROUBLESHOOTING .......................................................................................................... 9
APPENDIX 1: SAMPLE EQUILIBRATION WITH TUBE‐O‐DIALYZER™ (NOT SUPPLIED) ........ 10
APPENDIX 2: INSTRUCTIONS FOR CELL SURFACE PROTEIN BIOTINYLATION ................... 11
CELL SAMPLE PREPARATION:....................................................................................... 11
BIOTIN AGENT & CELL SURFACE REACTION ................................................................. 11
RELATED PRODUCTS ........................................................................................................ 11
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INTRODUCTION
This kit is designed for the conjugation of biotin to protein primary amine groups and is
supplied with the biotin reagent, a specific Optimizer Buffer™, for enhanced conjugation,
™
Spin‐OUT columns, for purification of labelled protein, and reagents to determine the
amount of biotinylation.
Biotin, a 244 Dalton molecule, exhibits an extraordinary binding affinity for avidin and
15
‐1
streptavidin (Ka=10 M ). The biotinylated molecules are efficiently probed with avidin
or streptavidin conjugated to reporter molecules, such as peroxidases or phosphatases.
The use of biotin labeled proteins in ELISA, Western blotting and dot blotting is a
popular technique.
The most widely used amine reactive biotinylation reagents are the water insoluble N‐
hydroxysuccinimide (NHS) esters or the water soluble N‐hydroxysulfosuccinimide (sulfo‐
‐
NHS) esters. The addition of a charged sulfonate (SO3 ) on the N‐hydroxysuccinimide
ring of the sulfo‐NHS esters results in their solubility in water (~10mM), but not
permeable to plasma membranes. The solubility and impermeability to plasma
membranes makes them ideal for studying cell surface proteins as they will only react
with the protein molecules on the outer surface of plasma membranes. The reactions of
the NHS and sulfo‐NHS esters with amines are virtually identical leading to the
formation of an amide bond and release of NHS or sulfo‐NHS.
™
HOOK Sulfo‐NHS‐LC‐Biotin kit is designed for the coupling of 1‐10mg protein in 1ml
buffer, suitable for 10 couplings.
Page 3 of 12
ITEMS SUPPLIED (Cat. # BS‐07)
Description
Size
HOOK™ Sulfo‐NHS‐LC‐Biotin Agent
25mg
Optimizer Buffer™ I [5X]
2 x 25ml
™
Spin‐OUT GT‐600, 5ml
10 columns
OneQuant™ HABA/Avidin
24 vials
™
BiotinQuant Assay Buffer
25ml
Biotin Standard
1ml
STORAGE CONDITION
The kit is shipped at Ambient Temp. Upon arrival, store the kit components at ‐20˚C.
Once the biotin reagent has been opened, store at ‐20°C with a desiccant as reagent is
moisture sensitive. Allow to warm to room temperature before opening.
SPECIFICATIONS
Molecular weight: 556.59
Spacer Arm (Å): 22.4
Membrane Permeable: No
Water Soluble: Yes
Reaction pH: 7‐9
PRECAUTIONS
™
1. Dissolve the HOOK Sulfo‐NHS‐LC‐Biotin immediately prior to use as the NHS‐ester
will hydrolyze and become inactive. Do not prepare stock solutions.
2. Avoid using primary amine containing buffers, such as Tris and Glycine, as these
will compete with the reaction.
ADDITIONAL ITEM(S) REQUIRED
15ml collection tubes
PREPARATION BEFORE USE
1. Dilute and prepare 1X Optimizer Buffer™ (1ml 5X Optimizer Buffer™ per 4ml de‐
ionized water).
2. Warm the Biotin‐Agent vial(s) to room temperature before opening to prevent the
condensation and deterioration of the biotin agent.
™
3. Add 50µl ultra pure water to a vial of OneQuant HABA/Avidin. Incubate at room
temperature for 5 minutes. Vortex to solubilize the HABA/Avidin.
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A. PROTEIN SAMPLE PREPARATION
™
1a Dissolve 1‐10mg protein in 0.5‐2ml 1X Optimizer Buffer I to a maximum
concentration of 10mg/ml.
1b If your protein is in an amine–free buffer at a pH of 7.2‐8.0 then proceed to the
next section.
1c For protein in Tris or other amine containing buffers a buffer exchange must be
™
performed. The buffer exchange can be done by dialysis against Optimizer Buffer
™
I, we recommend using our Tube‐O‐DIALYZER micro dialysis devices that ensure
™
no loss of precious protein (See Appendix 1). Or one of the supplied Spin‐OUT
columns can be used for buffer exchange as described in Section D. Please note
™
this kit is designed for 10 reactions and the Spin‐OUT columns are for purification
of the biotin labeled protein, using a column for buffer exchange will reduce the
number of reactions that can be performed. Additional columns can be ordered at
www.GBiosciences.com.
B. CALCULATION OF QUANTITY OF BIOTIN AGENT NEEDED FOR CONJUGATION
To achieve approximately 4‐6 biotin groups per antibody molecule, we recommend
using a 20 molar excess of biotin to antibody. The extent of biotin labeling for other
proteins is dependent on the distribution of amine groups and size of the protein,
therefore the molar ratio can be adjusted to suit your needs.
1. Millimoles of HOOK™ Sulfo‐NHS‐LC‐Biotin to be added for a 20 mole excess:
Protein Sample
™
Protein Sample
mmol HOOK Sulfo‐
X Concentration (mg/ml)
X 20 =
Volume (ml)
NHS‐LC‐Biotin
Protein Mol. Wt (Da)
2. µl 10mM HOOK™ Sulfo‐NHS‐LC‐Biotin to add:
500 µl HOOK™ Sulfo‐NHS‐LC‐Biotin
mmol HOOK™ Sulfo‐NHS‐LC‐
X 556.59 X
=
Biotin
solution
2.8
556.59= HOOK™ Sulfo‐NHS‐LC‐Biotin molecular weight
500 = µl of water 2.8mg of HOOK™ Sulfo‐NHS‐LC‐Biotin dissolved in for a 10mM solution
Example: For 0.5ml of a 5mg/ml IgG solution (150,000 Mol. Wt) solution.
5mg/ml
0.5ml X
X 20 = 0.000333mmol HOOK™ Sulfo‐NHS‐LC‐Biotin
150,000Da
™
500 33.1µl HOOK™ Sulfo‐NHS‐LC‐
0.000333mmol HOOK Sulfo‐NHS‐
X 556.59 X
=
LC‐Biotin
Biotin solution
2.8
C. PREPARATION OF BIOTIN AGENTS
1. Warm the biotin‐agent vials to room temperature before opening.
2. Immediately before using, add 500µl deionized water to every 2.8mg HOOK™ Sulfo‐
NHS‐LC‐Biotin for a 10mM working solution.
NOTE: Make fresh each time and do not prepare stock solutions.
Page 5 of 12
D. BIOTIN CONJUGATION REACTION
1. Add the calculated volume (Section B) of freshly prepared 10mM HOOK™ Sulfo‐
NHS‐LC‐Biotin to the protein solution from Section A.
2. Incubate the reaction at room temperature for 30‐60 minutes or on ice for 2 hours.
Longer incubations can be performed, however this may result in increased chance
of protein degradation.
E. REMOVAL OF UNCONJUGATED BIOTIN AGENTS
™
1. Prepare the Spin‐OUT column by removing the top and then bottom caps. Place
into a 15ml collection tube.
2. Centrifuge the column at 1,000g for 2 minutes to remove the storage buffer.
Discard storage buffer and return column to 15ml collection tube.
™
3. Equilibrate the column with 2ml 1X Optimizer Buffer I, by adding slowly to the
resin bed. Centrifuge at 1,000g for 2 minutes. Discard flow through and repeat this
step a further 2 times.
4. Place the column in to a clean 15ml collection tube and apply the sample directly to
the center of the resin bead. Allow the sample to migrate into the resin bed.
5. Centrifuge the column at 1,000g for 2 minutes. The flow through is the purified
labeled protein sample.
6. Store biotinylated protein at 4°C in 0.1% sodium azide until ready for use. Store at
‐20°C for long term storage.
F. ESTIMATION OF BIOTIN INCORPORATION EFFICIENCY
The method of biotin incorporation estimation is based on the binding of avidin with
HABA dye (2‐(4‐Hydroxyphenylazo)benzoic acid/ 2‐(4′‐Hydroxybenzeneazo)benzoic acid/
4′‐Hydroxyazobenzene‐2‐carboxylic acid), which produces a color that can be read at
500nm. The HABA‐avidin complex can be displaced with free biotin or biotin conjugated
with other molecules (proteins). Measuring the change in optical density of HABA‐avidin
complex with biotinylated proteins allows for accurate estimation of the molar ration of
biotin conjugated to the protein/ antibody.
Important Information
•
Ensure that all free/ unconjugated biotin is removed from the labeled protein or
other molecule before performing an estimation. We recommend desalting with
™
our SpinOUT desalting spin columns or dialysis with our micro dialysis devices,
™
Tube‐O‐DIALYZER .
•
During desalting or dialysis, we recommend exchanging the reaction buffer to
BiotinQuant™ Assay Buffer to ensure accurate estimation. PBS or TBS may also be
used, but avoid buffers containing potassium that may result in unwanted
precipitation.
•
A small variation in color between the OneQuant™ HABA/Avidin does not affect the
performance of the reagents.
Page 6 of 12
•
The Biotin Standard is supplied as an optional positive control for the assay. Use
100µl in lieu of the biotinylated sample. See calculation for determining amount of
biotin in the standard.
Protocol 1: Cuvette Protocol
1. Allow the reagents to warm to room temperature.
™
2. Pipette 850µl BiotinQuant Assay Buffer into a 1ml cuvette and zero the
spectrophotometer at a 500nm wavelength.
™
3. Briefly centrifuge a OneQuant HABA/Avidin vial and then transfer entire contents
to the cuvette and mix by gentle inversion.
4. Measure the absorbance of the HABA/Avidin complex at 500nm. This is your A500
HABA/Avidin reading.
5. Add 100µl biotinylated sample to the HABA/Avidin cuvette and mix well by
inversion.
NOTE: If using optional Biotin Standard, replace the 100µl biotinylated sample with
100µl Biotin Standard.
6. Measure the absorbance of the solution at 500nm. Record the absorbance once it
has stabilized for 10‐15 seconds. This is your A500 HABA/Avidin/Biotin Sample
reading.
NOTE: If the absorbance is <0.3, dilute the biotin sample and repeat the assay.
7. Go to the calculation section to determine the moles of biotin per mole of protein.
Protocol 2: Microplate Protocol
1. Allow the reagents to warm to room temperature.
™
2. Pipette 170µl BiotinQuant Assay Buffer into each microplate well. Blank the plate
™
reader with a well containing only BiotinQuant Assay Buffer.
™
™
3. Briefly centrifuge a OneQuant HABA/Avidin vial and then add 10µl OneQuant
HABA/Avidin to the cuvette and mix on an orbital shaker or equivalent.
4. Measure the absorbance of the HABA/Avidin complex at 500nm. This is your A500
HABA/Avidin reading.
5. Add 20µl biotinylated sample to the HABA/Avidin well and mix well as before.
NOTE: If using optional Biotin Standard, replace the 20µl biotinylated sample with
20µl Biotin Standard.
6. Measure the absorbance of the solution at 500nm. Record the absorbance once it
has stabilized for 10‐15 seconds. This is your A500 HABA/Avidin/Biotin Sample
reading.
NOTE: If the absorbance is <0.3, dilute the biotin sample and repeat the assay.
7. Go to the calculation section to determine the moles of biotin per mole of protein.
Page 7 of 12
Calculations
Based on Beer Lambert (Beer’s) Law: Aλ = ελbC, where
•
A is the absorbance at a particular wavelength (λ). HOOK™ BiotinQuant™ assay is
performed at 500nm.
•
ε is the extinction coefficient at the wavelength (λ). For HABA/Avidin samples at
500nm, pH7.0 this is
‐1
‐1.
34,000M cm .
•
b is the path length in centimeters. Cuvettes (10x10mm) have a pathlength of 1cm.
The pathlength for microplates, using the indicated volumes, is normally 0.5cm.
•
C is the molarity concentration of the sample (= mol/L = mmol/ml)
For calculating the number of moles of biotin per mole of protein or sample the
following values are required:
•
Concentration of protein/sample used (mg/ml)
•
Molecular weight of protein, expressed as grams per mole (e.g. IgG = 150,000)
•
A500 HABA/Avidin reading
•
A500 HABA/Avidin/Biotin Sample
•
Dilution factor (DF), if sample was diluted before adding to HABA/avidin solution.
1. Calculate mmol biotinylated protein/ml:
protein concentration (mg/ml)
Calculation #1:
=mmol protein/ml
MW of protein (mg/mmol)
2. Calculate change in absorbance at 500nm:
Calculation #2
(0.9 x A500 HABA/Avidin) –( A500 HABA/Avidin/Biotin
=ΔA500
Sample)
(Cuvette):
Calculation #2
(A500 HABA/Avidin) –( A500 HABA/Avidin/Biotin
=ΔA500
(Microplate):
Sample)
NOTE: 0.9 is the correction factor for the dilution of the HABA/Avidin with the sample in
the cuvettes. This is not necessary for microplates as the dilution is offset by the increase
in volume and therefore the light path (b).
3.
Calculate concentration of biotin in reaction (mmol/ml):
ΔA500
Calculation #2
mmol biotin
Calculation # 3:
=
=
34,000 x b
34,000 x b
ml reaction mixture
NOTE: b = lightpath, which is 1cm for cuvettes and 0.5cm for microplates.
4. Calculate mmol of biotin per mmol of protein:
mmol biotin in
Calculation #3 x
mmol biotin in
reaction x 10 x DF
10 x DF
Calculation # original sample
=
=
4:
mmol protein in
Calculation #1
Calculation #1
original sample
NOTE: DF is the dilution factor. 10 is for the 10 fold dilution of the biotinylated protein
sample in the reaction mixture.
Page 8 of 12
5.
Calculate concentration of biotin in Biotin Standard (mM):
ΔA500 x 10 x 1000
Calculation # 5:
= = [Biotin Standard] (mM)
34,000 x b
NOTE: b = lightpath, which is 1cm for cuvettes and 0.5cm for microplates. 10 is for the
10 fold dilution of the Biotin Standard in the reaction mixture.
TROUBLESHOOTING
Issue
Suggested Reason
Low or zero biotinylation of
protein.
Incomplete reagent mixing
ΔA500 is ≤0
Particulates in protein
solution interfering with
absorbance
Potassium ions present in
sample
Biotin levels are
unexpectedly high
Free, Unconjugated biotin
not removed
Possible Solution
Lack of functional groups for
biotinylation, use a different
coupling chemistry.
Ensure all the OneQuant™
HABA/Avidin is fully dissolved
before using
Filter protein solution before
assaying
Ensure samples are in
™
BiotinQuant Assay Buffer
Desalt or dialyze biotinylated
sample before use to remove
free biotin.
Page 9 of 12
APPENDIX 1: SAMPLE EQUILIBRATION WITH TUBE‐O‐DIALYZER™ (NOT
SUPPLIED)
If protein solution is in an incompatible buffer, dialyze and equilibrate into 1X Optimizer
™
Buffer as follows:
™
1. Pipette your sample directly into the Tube‐O‐DIALYZER tube. For Tube‐O‐
™
™
DIALYZER Micro use 20‐250µl and for Tube‐O‐DIALYZER Medi use 0.2‐2.5ml.
™
NOTE: Tube‐O‐Dialyzer is available in 1, 4, 8, 15 and 50kDa MWCO. Visit our
website for further information.
™
2. Pipette 3‐5ml appropriate 1X Optimizer Buffer into a Micro Dialysis Cup or small
beaker. If a small magnetic stir bar is available add to the Micro Dialysis Cup, if not
add 3‐5 glass balls.
™
3. Screw the dialysis cap on to the Tube‐O‐DIALYZER tube. Invert the Tube‐O‐
™
DIALYZER , ensuring the entire sample rests upon the membrane.
™
NOTE: If sample is too viscous, centrifuge the Tube‐O‐DIALYZER in an inverted
position (i.e. the dialysis membrane facing downward). Centrifuge for 5 seconds at
500‐1,000g.
™
4. Keeping the Tube‐O‐DIALYZER in an inverted position, slide the supplied float onto
™
™
the Tube‐O‐DIALYZER tube. Place the Tube‐O‐DIALYZER in the Micro Dialysis Cup
™
with the Optimizer Buffer .
5. Ensure that the dialysis membrane contacts the dialysis buffer. If there are large air
bubbles trapped underneath the dialysis membrane surface, tilt the tube or squirt
buffer to remove the air bubbles. Gently, stir the dialysis buffer with a magnetic stir
or place on an orbital shaker. For efficient and complete dialysis we recommend
™
inverting or gently tapping the Tube‐O‐DIALYZER 1‐2 times during dialysis to mix
the sample. If necessary repeat the centrifugation in step 3.
6. Dialyze at room temperature, or 4°C if required, for 1‐2 hours.
7. Repeat the dialysis with 1‐2 changes of buffer.
™
8. After dialysis, remove the Tube‐O‐DIALYZER from the float and immediately spin
™
the Tube‐O‐DIALYZER (in up‐right position) for 5‐6 seconds at 500‐1,000xg.
Page 10 of 12
APPENDIX 2: INSTRUCTIONS FOR CELL SURFACE PROTEIN BIOTINYLATION
™
NOTE: For cell surface protein labeling, we recommend our HOOK Cell Surface Protein
™
Isolation kit that uses G‐Biosciences HOOK biotin labeling and purification technology in
conjunction with our Mammalian Cell PE LB™ lysis buffer to conveniently label cell
surface proteins and isolate them for further analysis, including Western blotting.
Cell Sample Preparation:
1. Wash cells three times with ice‐cold PBS buffer to remove any contaminating
proteins.
6
2. Suspend the cells at a concentration of 25 x 10 cells/ml in PBS.
NOTE: Other cell concentrations can be used based on cell size, type, etc. The
concentration of biotinylation reagent can be scaled up or down accordingly.
Biotin Agent & Cell Surface Reaction
™
1. Prepare HOOK Sulfo‐NHS‐LC‐Biotin reagent by adding as described above 0.5mg
Biotin Agent per ml of reaction volume (solid form of biotin agent can be directly
added if it is water soluble).
2. Gently mix and incubate at room temperature for 30 minutes.
3. Wash cells three times with ice‐cold PBS to remove any remaining biotinylation
reagent. The cells surface proteins are now biotinylated.
RELATED PRODUCTS
Download our Protein Labeling & Conjugation Handbook
http://info.gbiosciences.com/complete‐protein‐labeling‐conjugation‐handbook/
For other related products, visit our website at www.GBiosciences.com or contact us.
Last saved: 7/10/2013 CMH
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