INSTRUCTIONS
FeBABE Protein Cutting Reagent
20332
1458.1
Number
Description
20332
FeBABE Protein Cutting Reagent, No-Weigh Format, 8 × 50 µg, cysteine-reactive (-SH group)
protein cutting reagent sufficient for eight labeling experiments
Br
Structure of FeBABE (right).
Chemical Name: Fe(III) (s)1-(p-bromoacetamidobenzyl)
ethylenediamine tetraacetic acid.
O
HN
H2O
Molecular Weight: 589.15
N
Labeling Group: Bromoacetyl
(labels sulfhydryl groups)
N
Fe+3
O
OH
O
Spacer Arm Length: 12 Å
O
O
O
O
O
Storage: Upon receipt store at 4°C. Product is shipped at ambient temperature.
Table of Contents
Introduction .................................................................................................................................................................................2
Glossary of Terms........................................................................................................................................................................2
Procedure Summary.....................................................................................................................................................................3
Important Product Information ....................................................................................................................................................3
Additional Materials Required.....................................................................................................................................................4
Procedure for FeBABE Conjugation and Activation...................................................................................................................4
A.
Removal of Free Metals from Protein ...........................................................................................................................4
B.
Conjugation of FeBABE Reagent to Cutting Protein....................................................................................................4
C.
Cutting/Activation Reaction ..........................................................................................................................................5
Troubleshooting...........................................................................................................................................................................6
Additional Information ................................................................................................................................................................6
Cited References..........................................................................................................................................................................7
General References......................................................................................................................................................................8
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Introduction
Protein:protein interaction sites can be mapped by targeted protein footprinting using the FeBABE Protein Cutting Reagent.
Targeted protein footprinting requires that two interacting proteins are first identified, isolated and sequenced. The protein of
interest (target protein) is either labeled at the N- or C- terminus with a radiolabel, fluorescent dye or an expression tag (e.g.,
polyhistidine, FLAG, C-myc) that can be detected by a specific antibody. Alternatively, an available antibody to an
endogenous N- or C-terminal epitope can also be used. The other interacting protein is then conjugated to FeBABE, an
EDTA-chelated iron atom linked to a sulfhydryl-reactive moiety, and becomes the cutting protein. The labeled target protein
and FeBABE-labeled cutting protein are allowed to interact; ascorbic acid and peroxide are then added to trigger the cutting
reaction. If the Fe-EDTA portion of the FeBABE-cutting protein is located near the contact site of the target protein, it will
cleave the polypeptide backbone of the target protein in proximity of the site. There is no cutting if the Fe-EDTA moiety is
oriented away from the contact site, or if it is inside the site and thus shielded from the activating reagents.1
The reaction mixture containing the cut protein complex is analyzed by gel electrophoresis or Western blot alongside two or
more specific chemical or enzymatic digests of the labeled target protein. The target protein is detected by the radiolabel or
fluorescent dye or through its terminal label or epitope. The resultant pattern represents only those fragments that extend
from the labeled end of the protein to the point of cleavage.2
Footprinting methods often use iron III chelated with EDTA that is added to a solution containing a protein:protein3 or
protein:DNA4 complex. The iron-chelated EDTA (Fe-EDTA) is then activated to produce hydroxyl radicals that
nonspecifically cut peptide or phosphodiester bonds in proximity. The cut fragments are analyzed by gel electrophoresis or
Western blot, and the pattern is compared with patterns from Fe-EDTA generated fragments of the individual proteins
involved in the interaction. However, fragment patterns from Fe-EDTA in solution can be complex and difficult to interpret.
Using FeBABE to tether Fe-EDTA metal chelates to the cutting protein at known sites results in fewer fragments than using
free Fe-EDTA in solution, simplifying analysis. 5 This tether method provides greater signal-to-noise ratio for better
sensitivity, which is particularly valuable for weak interactions. FeBABE enables labeling of the cutting protein at known
sites based on the protein sequence. The FeBABE Protein Cutting Reagent can be especially useful for mapping the
interaction sites for multi-protein complexes where crystallization is not possible.6
Glossary of Terms
Bait Protein: see cutting protein
Cutting protein: the protein that is conjugated to FeBABE.
FeBABE: an EDTA-chelated iron atom linked to a sulfhydryl-reactive moiety or Fe(III) (s)-1-(p-Bromoacetamidobenzyl)
ethylenediaminetetraacetic acid.
Footprinting: the process of mapping the site at which a protein binds to DNA, RNA or another protein by comparing the
fragmentation patterns generated by chemical or enzymatic cleavage of the bound complex and of the individual proteins.
Interaction site: the location on the target protein where contact occurs with the cutting protein.
Prey protein: see target protein
Target protein: the protein that is being studied; it must be known to interact with the cutting protein.
Targeted protein footprinting: conjugating the metal chelate complex to be used in footprinting to known sites on the
cutting protein.
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Procedure Summary
1. Treat cutting protein with Metal
Removal Reagent. Incubate for 24 hours.
2. Exchange cutting protein into
Conjugation Buffer.
3. Reconstitute contents of one tube of the
FeBABE Protein Cutting Reagent.
4. Add FeBABE to cutting protein.
Incubate for 1 hour at 37°C.
5. Exchange FeBABE-cutting
protein conjugate into Cutting
Buffer.
6. Mix target protein with FeBABEcutting protein conjugate. Incubate at
room temperature for 30 minutes.
Ascorbic Acid
Peroxide
8. Activate FeBABE by rapid sequential
addition (i.e., ≤5 seconds) of ascorbic acid
and peroxide. Incubate for 30 seconds.
Sample Buffer + Glycerol
9. Stop reaction by adding electrophoresis
sample buffer mixed with glycerol. Analyze
results by SDS-PAGE and/or Western blot.
Important Product Information
To use the FeBABE Protein Cutting Reagent mapping technology, the following conditions must be met:
•
The two interacting proteins being evaluated must be purified and of known sequence.
•
The target protein must be suitable for end labeling with a fluorescent or radiolabel tag. Alternatively, an antibody
specific for the N- or C-terminal amino acid epitope must be available. Antibodies to expression tags, such as
polyhistidine, FLAG or C-myc, also may be used.
•
The cutting protein must have at least one sulfhydryl that can react with the bromoacetyl moiety of FeBABE. The
sulfhydryl may be either endogenous or cloned cysteine mutants. Alternatively, 2-iminothiolane (Traut’s Reagent,
Product No. 26101) can be used to randomly modify an average of 1-2 lysine residues to sulfhydryls. Sulfhydryls can be
verified using Ellman’s Assay (Product No. 22582).
Note: Modifying the cutting protein using Traut’s Reagent is sometimes used as a screening method to locate the general
area of the interaction.7,8
•
Peptide fragments of the target protein generated by site-specific chemical or enzymatic cleavage are necessary for use
as Western blot mobility or cleavage standards. Molecular weight markers may be used for an initial size approximation;
however, accurate determination of the residues involved in the binding site requires comparison of sample cleavage
bands to proteolytic fragments of the target protein. (See Table 2 in the Additional Information section for a partial list of
cleaving reagents.)
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Additional Materials Required
Note: Use low metal content components in reagent formulations to minimize nonspecific cutting of the target protein.
•
Metal Removal Buffer: 30 mM MOPS, 4 mM EDTA, pH 8.2
•
Conjugation Buffer: 30 mM MOPS, 100 mM NaCl, 1 mM EDTA, 5% glycerol; pH 8.2
•
Cutting buffer: 50 mM MOPS, 120 mM NaCl, 0.1 mM EDTA, 10 mM MgCl2, 10% glycerol; pH 8.0
•
Ascorbic Acid: Immediately before use (see step C4) prepare a solution containing 40 mM ascorbic acid, 10 mM EDTA,
pH 7.0
•
Hydrogen Peroxide: Immediately before use (see step C4) prepare a solution containing 40 mM hydrogen peroxide,
10 mM EDTA
•
Electrophoresis sample loading buffer mixed 1:1 with 80% glycerol
Note: Sample loading buffer with glycerol is used to stop the cutting reaction. Glycerol serves as a free radical scavenger
and halts the cutting action of the reduced Fe-EDTA. Although most sample loading buffers contain glycerol, increasing
the glycerol content limits nonspecific cleavage after the desired cuts are made.
•
Traut’s Reagent (2-Iminothiolane, Product No. 26101) − see Important Product Information section to determine if
Traut’s reagent is required
•
Variable-speed bench-top microcentrifuge
•
Two pipettors capable of accurately measuring 1-10 µl
•
0.5 and 1.5 ml microcentrifuge tubes
•
Desalting spin columns (e.g., Product No. 89849) or a dialysis product (e.g., Slide-A-Lyzer® MINI Dialysis Unit Plus
Float, Product No. 69576; or Slide-A-Lyzer Dialysis Cassette Kit, Product No. 66385)
Procedure for FeBABE Conjugation and Activation
Equilibrate FeBABE Reagent to room temperature. Keep reagent in foil pouch until ready to use.
A. Removal of Free Metals from Protein
1.
Dissolve cutting protein in metal removal reagent. Use a final protein concentration of 5-50 nmol/ml. If sample is
already in a buffer, dialyze or buffer exchange the sample into the metal removal buffer. Incubate at 4°C in the metal
removal buffer for at least 24 hours before proceeding.
2.
Buffer exchange sample into conjugation buffer using a desalting column or dialysis.
Note: Save some protein solution for use as a control for the Western blot analysis.
3.
Determine the protein concentration using a suitable protein assay.
Note: The protein may be stored in conjugation buffer at -20°C or -80°C or proceed directly to Section B.
B. Conjugation of FeBABE Reagent to Cutting Protein
1.
Calculate amount of FeBABE Reagent needed for conjugation. For best results, use 2- to 20-fold molar excess of
FeBABE to moles of free sulfhydryl on the cutting protein. For initial trials, use a 20-fold molar excess.
Note: After reconstitution, the concentration of FeBABE is 8.5 nmol/µl (5 mg/ml) in 10 µl total volume (589 MW).
2.
If Traut’s Reagent will be used to add sulfhydryls to the cutting protein, prepare a solution containing ~0.5 mg/ml
(3.6 nmol/µl) in conjugation buffer. Calculate the amount of Traut’s Reagent needed to equal half the moles of FeBABE
in the final conjugation reaction.
3.
Use an empty pipette tip to puncture the foil covering of one No-Weigh Microtube of FeBABE Reagent. Add 10 µl of
Conjugation Buffer to one microtube, which results in 8.5 nmol/µl (5.0 mg/ml) solution.
4.
Gently mix by drawing liquid up and down in the pipette tip several times. The reagent usually dissolves in less than 30
seconds.
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Note: Use reconstituted FeBABE Cutting Reagent immediately. Discard the used tube by cutting it from the remaining
unused tubes. Return unused microtubes to foil pouch with desiccant pack, seal securely and store at 4°C.
5.
Add required amount of FeBABE solution calculated in step B1 to cutting protein in a 1.5 ml microtube. If desired,
immediately add Traut’s Reagent to a final amount that is equal to half the moles of FeBABE in the reaction.
6.
Cover sample tube with foil to exclude light and incubate at 37°C for 1 hour. Mix during reaction if possible.
7.
After the 1 hour incubation, immediately exchange sample into Cutting Buffer using a desalting spin column or a
dialysis cassette.
8.
Remove 5-10 µl of the conjugate (depending on sample volume) for evaluation by protein assay and immediately place
the FeBABE-protein conjugate at -80°C.
9.
Determine protein concentration by protein assay.
Note: Immediately proceed to Section C, or store the desalted FeBABE-protein conjugate in Cutting Buffer at -80°C.
For extended storage (i.e., >1 week) adjust glycerol content to 50%.
C. Cutting/Activation Reaction
1.
Buffer exchange the target protein into the Cutting Buffer.
2.
Mix FeBABE-protein conjugate with the target protein at equal molar ratios and allow the two proteins to interact at
room temperature for 30 minutes.
3.
This protocol uses 50-300 pmol FeBABE-cutting protein in 20 µl of Cutting Buffer. If other sample sizes are used, adjust
reaction volumes to keep the ratios of sample:Ascorbic Acid and sample:Hydrogen Peroxide at 8:1 each.
4.
Prepare a solution of Ascorbic Acid containing 40 mM ascorbic acid, 10 mM EDTA, pH 7.0. Also prepare a solution of
Hydrogen Peroxide containing 40 mM hydrogen peroxide, 10 mM EDTA.
5.
Perform FeBABE-protein cutting reaction on the sample(s) as described in the following steps (i.e., Steps 14a-f).
Complete the cutting reaction on one sample before beginning another.
a)
b)
c)
d)
e)
f)
6.
Adjust sample concentration as described in Step C3.
Add 2.5 µl of Ascorbic Acid to sample. Immediately vortex for 2-3 seconds.
IMMEDIATELY add 2.5 µl of Hydrogen Peroxide and vortex for 2-3 seconds.
Incubate reaction mixture for 30 seconds from the time the Hydrogen Peroxide is added.
Add 25 µl of sample loading buffer with glycerol to stop reaction.
Store the cut FeBABE-protein-protein sample at -80°C.
After the FeBABE-protein-protein samples are cut, prepare controls. Suggested controls are listed in Table 1. Prepare the
first two controls (i.e., target protein only and cutting protein + target protein; no FeBABE) each time the cutting
reaction is performed. Always dilute protein to the same concentration as used in the cutting reaction.
Note: Immediately proceed to analysis by gel electrophoresis and/or Western blot (see Additional Information section
for analysis information). Alternatively, store in sample loading buffer at -80°C.
Table 1. Suggested controls for gel electrophoresis and/or Western blot. Controls are processed through the cutting
reaction if the table indicates addition of both ascorbic acid and stable peroxide buffer. The remaining controls are diluted
with cutting buffer in place of ascorbic acid and stable peroxide buffer before adding the sample loading buffer.
Control Sample
Cutting
Buffer
Ascorbic
Acid
Stable
Peroxide
Reagent
SDS Sample
Loading Buffer
with Glycerol
X
X
X
X
*Target protein only
*Cutting protein + target protein; no FeBABE
X
X
X
X
**Target Protein + FeBABE-labeled non-relevant protein
X
X
X
X
Target Protein only
X
X
Target protein mobility standards
X
X
*Prepare this control each time the cutting reaction is performed.
**Optional control for confirmation of interaction specificity.
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Troubleshooting
Problem
Possible Cause
Western blot has bands for
controls and intact target protein,
but no bands for fragments
FeBABE is conjugated to cutting protein
at a position oriented away from the
contact site or at a position that is
interfering with the interaction, or it is
inside the site and thus shielded from the
activating reagents
If target protein is from a recombinant
source, add cysteine to a different location
There are no free sulfhydryls on cutting
protein and, therefore, no conjugate was
formed
Test protein for sulfhydryl content using
Ellman’s reagent
Sample was nonspecifically cut by iron
contaminants
Increase time in Metal Removal Buffer and
check for other sources of iron contamination
then repeat FeBABE conjugation
There is a smear of bands on the
Western blot
Solution
React cutting protein with Traut’s Reagent
(2-iminothiolane) during the conjugation
reaction and repeat cutting
React cutting protein with Traut’s Reagent
(2-iminothiolane) during the conjugation
reaction and repeat cutting
Eliminate heating step before electrophoresis
No bands are present on the
Western blot
Western blot not optimized
Optimize blot using intact proteins and
control samples
Additional Information
A. Preparation of Molecular Weight Standards
Based on the known sequence of the target protein, select at least two specific digestion methods to generate molecular
weight markers (mobility standards) of the cut sample. Choose digestion methods that will result in fragments encompassing
the entire molecular weight range of the target protein. See Table 2 for a partial list of chemical and enzymatic cleavage
agents.
Table 2. Chemical and enzymatic cleavage agents for preparing target protein mobility standards.
Cleavage Site
Cleavage Agent
Expected Results
Tryptophan
(C-terminal)
Cyanogen Bromide
(after oxidation)
Large fragments
9
Methionine
(C-terminal)
Cyanogen Bromide
(after reduction)
Large fragments
9
Cysteine
(N-terminal)
NTCB
(2-Nitro-5-thiocyanobenzoate)
Large fragments
10
Asparaginyl-glycyl
peptide bonds
Hydroxylamine
(Product No. 26103)
Large fragments
11
Lysines and Arginine
(C-terminal)
Trypsin
(Product No. 20233)
Small fragments
Lysine
(C-terminal)
Endoproteinase Lys-C
*Glutamic acid or Glutamic and
Aspartic Acids (C-terminal)
Endoproteinase Glu-C
(S. aureus V8, Product No. 20195)
Large fragments
Arginine
(C-terminal)
Submaxillaris Protease
(Product No. 20199)
Large fragments
15
Arginine
(C-terminal)
Clostripain
(Endoproteinase Arg-C)
Large fragments
16
Large fragments
12
13,14
3
*Cleavage site depends on reaction conditions.
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B. Western Blot
Once the cutting reaction has been completed, analyze the sample, controls and mobility standards by Western blot. For the
initial trial of a new protein, use several dilutions of the cut sample to determine the detection range for the specific system.
For the closest possible estimate of the cut site(s) location, also process the sample, controls and standards on gels with
differing molecular weight separation ranges.
C. Analysis
Scan the Western blot and analyze using densitometry software. Plot a standard curve of migration distance vs. amino acid
residue number using the protein digest mobility standards. This plot is used to provide a better estimate than can be
accomplished visually of which target protein amino acids are adjacent to the interaction site. While analyzing bands, be
aware of the following points:
• Band intensity is related to how accessible the Fe-EDTA moiety of FeBABE is to the cutting reagents in the surrounding
buffer. If FeBABE is very close to the interaction site with the target protein, the ascorbic acid and peroxide will not be
readily available for reaction with Fe-EDTA. The result will be a weak band on the Western blot. If the Fe-EDTA moiety of
FeBABE is a few residues further away from the target contact site, it will be more accessible to the cutting reagents and the
band on the Western blot will be more intense.
• There is a possibility that the fragments generated in the cutting reaction may themselves be cut if the cutting reaction is
not optimized. These “fragments of fragments” result in extra bands on the Western blot, and it becomes difficult to
determine if a band is from cutting the target protein or from cutting a fragment. Examine the Western blot to determine
relative intensity of the fragment band(s) in the cut sample relative to the uncut target protein control. If the fragment band is
greater than approximately 10% of the target protein band, there is a chance that some fragments have been cut twice. If cuts
of fragments are suspected, optimize the cutting reaction by varying cutting time, amount of protein-protein complex and the
ascorbic acid and peroxide concentration ratios to protein concentration. Bands that are the result of multiple cuts will not
appear with less cutting time, while bands that are the result of cuts near the binding site will be present.
Related Thermo Scientific Products
32223
26101
24590
88024
34080
34090
FeBABE Protein Interaction Mapping Kit
2-Iminothiolane-HCl, 500 mg
GelCode® Blue Stain Reagent, a total protein stain for SDS-PAGE, 500 ml
Nitrocellulose Membrane (8 cm x 8 cm), 15 sheets/pkg
SuperSignal® West Pico Chemiluminescent Substrate for HRP,** 500 ml
CL-XPosure™ Film (5” x 7”), 100 sheets
Cited References
1.
Datwyler, S.A. and Meares, C.F. (2001). In Metal Ions in Biological Systems, Volume 38, Sigel, A. and Sigel, H., Eds. (Marcel Dekker, New York)
pp. 213-54.
2.
Miyake, R., et al. (1998). Dimeric association of Escherichia coli RNA polymerase α subunits, studied by cleavage of single-cysteine α subunits
conjugated to iron-(S)-1-[p-(Bromoacetamido)benzyl]ethylenediaminetetraaccetate. Biochemistry 37:1344-49.
3.
Heyduk, T., et al. (1996). Determinants of RNA polymerase α subunit for interaction with β,β´ and σ subunits; Hydroxyl-radical protein footprinting.
Proc. Natl. Acad. Sci. USA 93(101):62-6.
4.
Gross, P., et al. (1998). Hydroxyl radical footprinting of DNA complexes of the ets domain of PU.1 and its comparison to the crystal structure.
Biochemistry 37(15):5129-35
5.
Datwyler, S.A. and Meares, C.F. (2000). Protein-protein interactions mapped by artificial proteases: where σ factors bind to RNA polymerase. TIBS
25:408-14.
6.
Wigneshweraraj, S.R., et al. (2000). Conservation of sigma-core RNA polymerase proximity relationships between the enhancer independent and
enhancer-dependent sigma classes. EMBO J. 19:3038-48.
7.
Traviglia, S.L., et al. (1999). Targeted protein footprinting; where different transcription factors bind to RNA polymerase. Biochemistry 38:15774-8.
8.
Traviglia, S.L., et al. (1999). Mapping protein-protein interactions with a library of tethered cutting reagents: The binding site of σ70 on Escherichia
coli RNA polymerase. Biochemistry 38:4259-65.
9.
Smith, B.J. (1994). In Basic Protein and Peptide Protocols, Walker, J.M., Ed. (Humana Press, Totowa, New Jersey) 297-309.
10. Smith, B.J. (1996). In The Protein Protocols Handbook, Walker, J.M., Ed. (Humana Press, Totowa, New Jersey) 385-7.
11. Smith, B.J. (1996). In The Protein Protocols Handbook, Walker, J.M., Ed. (Humana Press, Totowa, New Jersey) 389-92.
12. Smith, B.J. (1994) in Basic Protein and Peptide Protocols, Walker, J.M., Ed. (Humana Press, Totowa, New Jersey) 289-296.
Pierce Biotechnology
PO Box 117
(815) 968-0747
3747 N. Meridian Road
Rockford, lL 61105 USA
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13. Dong, M., et al. (1999). Demonstration of a direct interaction between residue 22 in the carboxyl-terminal half of secretin and the amino-terminal tail
of the secretin receptor using photoaffinity labeling. J. Biol. Chem. 274:903-9.
14. Phalipou, S., et al. (1997). Mapping peptide-binding domains of the human V1a vasopressin receptor with a photoactivatable linear peptide
antangonist. J. Biol. Chem. 272:26536-44.
15. Moonen, P., et al. (1980). The primary structure of the phosphatidylcholine-exchange protein from bovine liver. Eur. J. Biochem. 106:279-290.
16. Mitchell, W.M., and Harrinton, W.F. (1968). Purification and properties of clostridiopeptidase B (Clostripain). J. Biol. Chem. 243:4683-92.
General References
Ghaim, J.B., et al. (1995). Proximity mapping the surface of a membrane protein using an artificial protease: Demonstration that the quinone-binding
domain of subunit I is near the N-terminal region of subunit II of cytochrome bd. Biochemistry. 34:11311-15.
Lee, J., et al. (2000). Phosphorylation-induced signal propagation in the response regulator. J. Bacteriol. 182:5188-95.
Olekhnovich, I.N. and Kadner, R.J. (2002). Mutational scanning and affinity cleavage analysis of UhpA-binding sites in the Escherichia coli uhpT
promoter. J. Bacteriol. 184(10):2682-91.
Owens, J.T., et al. (1998). Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease. Proc. Natl.
Acad. Sci. USA 95:6021-26.
Rana, T.M. and Meares, C.F. (1991). Transfer of oxygen from an artificial protease to peptide carbon during proteolysis. Proc. Natl. Acad. Sci. USA.
88:10578-82.
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