INSTRUCTIONS
Pierce™ S-Nitrosylation
Western Blot Kit
Online Specials
90105 90106
2464.1
Number
Description
90105
Pierce S-Nitrosylation Western Blot Kit, sufficient reagents for 40 reactions
HENS Buffer, 100mL, store at room temperature
Methyl Methanethiosulfonate (MMTS), 200mg, store at 4°C
Sodium Ascorbate, 20mg, store at room temperature
iodoTMTzero™ Label Reagent, 2 × 0.2mg, store at -20°C
Anti-TMT™ Antibody, 100µL, store at -20°C
90106
HENS Buffer, 100mL
Contents: 100mM HEPES, pH 7.8; 1mM EDTA; 0.1mM Neocuproine; 1% SDS
Storage: Upon receipt store kit at -20°C or store individual components as indicated above. Kit is
shipped with an ice pack. Store HENS Buffer at room temperature. HENS Buffer is shipped at ambient
temperature.
Introduction
The Thermo Scientific Pierce S-Nitrosylation Western Blot Kit enables detection of protein S-nitrosocysteine posttranslational modifications. This kit provides all of the necessary reagents for a modified S-nitrosylation switch assay, but
uses a non-biological iodoTMT™ Reagent for labeling instead of HPDP-biotin for less background during Western blot
detection. Similar to the traditional S-nitrosylation switch assay, unmodified cysteines are first blocked using a sulfhydrylreactive compound (e.g., MMTS). S-nitrosylated cysteines are then selectively reduced with ascorbate in HENS Buffer for
specific labeling with iodoTMTzero Reagents, which irreversibly bind to the cysteine thiol that was S-nitrosylated (Figure 1).
Detection of the TMT reagent-modified proteins is facilitated using an anti-TMT antibody. In addition to detection of Snitrosylated proteins by Western blot, an immobilized anti-TMT resin can be used to selectively enrich S-nitrosylated
proteins/peptides labeled with iodoTMT reagents. This workflow allows for S-nitrosylation site mapping and multiplexed
quantititation using mass spectrometry.
Figure 1. Workflow for the Thermo Scientific Pierce S-Nitrosylation Western Blot Kit. S-nitrosylated protein
samples are first reacted with MMTS to block free sulfhydryls. S-nitrosocysteines are selectively reduced with
ascorbate for labeling with the iodoTMTzero reagent. Finally, the anti-TMT antibody is used for Western blot
detection of the TMT-labeled proteins.
Pierce Biotechnology
PO Box 117
(815) 968-0747
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
www.thermoscientific.com/pierce
Important Product Information
•
Thaw HENS Buffer using a room temperature water bath and mix to resuspend. Presence of a precipitate does not
adversely affect HENS Buffer performance.
•
MMTS is a liquid reagent (~99% pure = 10.6M solution) with a very strong odor. Use a syringe needle to transfer small
reagent volumes in a fume hood to make stock solutions.
•
MMTS has poor solubility when added directly to aqueous buffers. Dilute MMTS 1:10 with DMF before addition to
samples and mix vigorously by vortexing before and after addition to samples to ensure complete free thiol blocking.
•
Stock solutions of sodium ascorbate are unstable. Make new solutions for each procedure.
•
iodoTMT Reagents are light- and free radical-sensitive. Minimize exposure to light during labeling reactions by
wrapping reaction tubes in aluminum foil or placing reactions in a space protected from light. Avoid using water or
buffers containing reduced metals (e.g., copper).
•
S-nitrosocysteine modifications are highly labile. Minimize exposure to light during sample preparation until Labeling
Reagent addition. Avoid the use of strong reducing agents (e.g., DTT and TCEP) and metal ions in water and buffers.
•
Perform all incubations and centrifugation steps at room temperature unless otherwise noted.
•
For best results when performing the Western blotting procedure, use Thermo Scientific Pierce Goat Anti-Mouse IgG
(H+L), Peroxidase Conjugated (Product No. 31430) and Thermo Scientific SuperSignal West Pico Chemiluminescent
Substrate (Product No. 34080). If similar products from other vendors are used, the Western blotting procedure must be
optimized.
•
Removal of non-reacted iodoTMT Label Reagent is required for successful enrichment of labeled proteins or peptides
using Anti-TMT Resin (Product No. 90076), but is not required for Western blot detection. Strategies for removal of the
label reagent are found in Tech Tip #69 on our website.
Additional Materials Required
•
Ultrapure water
•
Methanol or Dimethylsulfoxide (DMSO), Sequencing Grade (Product No. 20688)
•
Dimethylformamide (DMF), Sequencing Grade (Product No. 20673)
•
Acetone, chilled (-20°C)
•
Pierce BCA Protein Assay Kit (Product No. 23227)
•
Polyacrylamide gel, 12% or 4-20% (Thermo Scientific Precise Protein Gels; see catalog or website)
•
•
Nitrocellulose (Product No. 88014) or PVDF (Product No. 88585) membrane
Nonfat dry milk (NFDM) or nonfat dry milk with tris-buffered saline (TBS) (Thermo Scientific Blocker Blotto Blocking
Buffer, Product No. 37530)
•
Pierce Goat Anti-Mouse IgG-Horseradish Peroxidase Conjugate (Product No. 31430)
•
20X TBS Tween™-20 Buffer (TBST) (Product No. 28360)
•
SuperSignal™ West Pico Chemiluminescent Substrate (Product No. 34080)
•
X-ray film (Thermo Scientific CL-XPosure Film (Product No. 34090 or 34091) or a CCD camera
•
Optional: Reduced glutathione (Product No. 78259)
•
Optional: S-nitrosoglutathione
Pierce Biotechnology
PO Box 117
(815) 968-0747
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
2
www.thermoscientific.com/pierce
S-Nitrosylated Protein Labeling and Western Blot Detection
Note: Use 100-200µg protein per sample. Use S-nitrosoglutathione to generate a control sample for method optimization.
Material Preparation
1M MMTS
Dilute 20μL of MMTS with 180μL of DMF in fume hood.
1M Sodium ascorbate
Dissolve 20mg of sodium ascorbate with 0.1mL of ultrapure water.
20mM Labeling Reagent
Dissolve 0.2mg iodoTMT Reagent with 20μL of methanol or DMSO.
Note: Labeling Reagent stock solutions can be stored at -20°C.
A. Protein Extraction, Blocking and S-nitrosocysteine Labeling
1.
Culture cells to harvest at least 100μg of protein per condition. For best results, culture at least 5 × 106 cells.
2.
Lyse cells with 4 cell-pellet volumes of HENS Buffer (i.e., use 4mL of HENS Buffer per milliliter of cells).
Note: Sonicate lysates to reduce viscosity.
3.
Centrifuge sample at 10,000 × g for 10 minutes.
4.
Optional: Incubate sample with 200μM of S-nitrosoglutathione for 30 minutes at room temperature to generate a control
sample. Use reduced glutathione to generate a negative control. Remove unreacted S-nitrosoglutathione using Thermo
Scientific Zeba Spin Desalting Columns pre-equilibrated in HENS Buffer.
5.
Perform a protein assay (e.g., BCA Protein Assay) to determine the protein concentration.
6.
Prepare protein at 1-2mg/mL in at least 100µL of HENS Buffer. Use 100µg per condition. Ensure the protein
concentration is equal for each sample.
7.
To each 100µL sample, add 2μL of 1M MMTS (20mM final concentration), vortex vigorously for 1 minute to mix and
incubate for 30 minutes at room temperature to block free cysteine thiols.
8.
Precipitate protein by adding six volumes (e.g., 600μL) of pre-chilled (-20°C) acetone and freezing at -20°C to remove
MMTS. Allow the precipitation to proceed for at least 1 hour.
Note: Removal of excess MMTS is required before iodoTMT Reagent labeling. In addition to acetone precipitation, MMTS
can be removed using Zeba™ Spin Desalting Columns pre-equilibrated in HENS Buffer.
9.
Centrifuge the samples at 10,000 × g for 10 minutes at 4°C. Carefully invert the tubes to decant the acetone without
disturbing the white pellet. Allow the pellet to dry for 10 minutes.
10. Resuspend precipitated samples in 100μL of HENS Buffer and divide the sample into two new microcentrifuge tubes.
11. Add 1μL of the Labeling Reagent to each 50μL of sample and briefly vortex to mix.
12. Add 2μL of 1M sodium ascorbate to each sample and briefly vortex to mix.
Note: For negative control reactions, add 2μL of ultrapure water instead of sodium ascorbate.
13. Allow the reaction to proceed for 1-2 hours at room temperature.
Optional: Add six volumes (~600μL) of pre-chilled (-20°C) acetone and freeze at -20°C. Allow the precipitation to
proceed for at least 1 hour. Remove acetone and allow samples to dry for 10 minutes. Resuspend with 100μL of HENS
Buffer.
B. Labeled-Protein SDS-PAGE and Western Blotting
Note: This procedure has been optimized using SuperSignal West Pico Chemiluminescent Substrate (see Important
Product Information Section). Perform all blocking, probing and washing incubation steps using constant agitation.
1.
Add 10μL of 5X reducing Laemmli sample buffer to 40μL of labeled sample. Heat the eluted sample for 5 minutes at
95-100°C.
2.
Separate proteins by SDS-PAGE. Apply at least 25µL per lane for a 10 × 10cm mini-gel.
Pierce Biotechnology
PO Box 117
(815) 968-0747
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
3
www.thermoscientific.com/pierce
3.
Transfer to nitrocellulose or PVDF membrane.
4.
Block membrane in 5% NFDM in TBST for 1 hour.
5.
Prepare the Anti-TMT Antibody (1:1000) in 5% NFDM in 1X TBST.
6.
Incubate the membrane in the primary anti-TMT antibody solution for 1 hour.
7.
Wash the membrane five times for 5 minutes each with 1X TBST.
8.
Dilute the anti-mouse IgG-HRP conjugate in 5% NFDM in 1X TBST [e.g., if using Pierce Goat Anti-Mouse IgG (H+L),
Peroxidase Conjugated, dilute within 1:20,000 to 1:100,000].
9.
Incubate membrane in the anti-mouse IgG-HRP conjugate solution for 1 hour at room temperature.
10. Wash the membrane five times for 5 minutes each with 1X TBST.
11. Incubate membrane with SuperSignal West Pico Chemiluminescent Substrate for 5 minutes.
12. Immediately expose the membrane to X-ray film or an imager (Thermo Scientific MYECL Imager, Product No. 62236).
Troubleshooting
Problem
S-nitrosocysteine
signal not detected
Possible Cause
Sodium ascorbate or Labeling
Reagent were not added
Incomplete removal of MMTS
Solution
Add Labeling Reagent followed by sodium ascorbate to
selectively reduce S-nitrosylated cysteines for detection
Rinse acetone pellets with ice-cold water
Use Zeba Spin Desalting Columns instead of acetone
precipitation
Protect samples from light until Labeling Reagent addition
Avoid reducing agents during sample prep
Decrease total sample handling time by using Zeba Spin
Desalting Columns instead of acetone precipitation
Empirically determine the optimal primary antibody
concentration
Use an S-nitrosocysteine donor such as S-nitrosoglutathione
as a positive control
Use goat anti-mouse IgG HRP conjugates
S-nitroso group was labile
Primary antibody required
optimization
S-nitroso levels were too low
Western blot resulted
in high background
Incorrect secondary antibody was
used for detection
Free sulfhydryls were not
completely blocked
Vigorously mix MMTS reagent with sample during
blocking reactions
Increase MMTS concentration, incubation time and/or
temperature
Consult instructions for the HRP substrate being
used
Inadequate blot blocking or
washing
Secondary antibody concentration
was too high
Additional Information Available on Our Website
•
Tech Tip #49: Acetone precipitation of proteins
Pierce Biotechnology
PO Box 117
(815) 968-0747
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
4
www.thermoscientific.com/pierce
Related Thermo Scientific Products
90075
Anti-TMT Antibody, 100μL
90076
Immobilized Anti-TMT Resin, 6mL
90100
90102
iodoTMTzero Label Reagent Set, 5 × 0.2mg
iodoTMTsixplex™ Isobaric Label Reagent Set, 1 × 0.2mg
90103
iodoTMTsixplex Isobaric Mass Tag Labeling Kit, 5 × 0.2mg
90104
TMT Elution Buffer, 20mL
89890
Zeba Spin Desalting Columns, 2mL, 5 units
23011
MMTS, 200mg
78259
Glutathione, 5 × 184mg
23030
N-Ethylmaleimide (NEM), 25g
21341
EZ-Link™ HPDP-Biotin, 50mg
28360
20X TBS Tween-20 Buffer, 500mL
31430
Pierce Goat Anti-Mouse IgG (H+L), Peroxidase Conjugated, 2mL
34079
SuperSignal West Pico Chemiluminescent Substrate, 500mL
34090
CL-XPosure™ Film (5" × 7"), 100 sheets/pkg
88014
Nitrocellulose Membrane, 0.45µm, 7.9cm × 10.5cm
88585
PVDF Membrane, 0.45µm, 7.9cm × 10.5cm
46430
Restore™ Plus Western Blot Stripping Buffer, 500mL
62236
MYECL™
Imager
General References
Jaffrey, S.R., et al. (2001). The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE. 86:p11.
Murray, C.I. (2012). Identification and quantification of S-nitrosylation by cysteine reactive tandem mass tag switch assay. Mol Cell Proteomics
11(2):M111.013441.
Tween is a trademark of Croda International PLC.
ICAT is a trademark of the University of Washington.
iTRAQ is a trademark of Life Technologies Corp.
TMT, iodoTMT, iodoTMTzero and iodoTMTsixplex are trademarks of Proteome Sciences PLC.
Products are warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in
the Product documentation, specifications and/or accompanying package inserts (“Documentation”). No claim of suitability for use in applications regulated
by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this
warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to
anyone other than Buyer. Any model or sample furnished to Buyer is merely illustrative of the general type and quality of goods and does not represent that
any Product will conform to such model or sample.
NO OTHER WARRANTIES, EXPRESS OR IMPLIED, ARE GRANTED, INCLUDING WITHOUT LIMITATION, IMPLIED WARRANTIES OF
MERCHANTABILITY, FITNESS FOR ANY PARTICULAR PURPOSE, OR NON INFRINGEMENT. BUYER’S EXCLUSIVE REMEDY FOR NONCONFORMING PRODUCTS DURING THE WARRANTY PERIOD IS LIMITED TO REPAIR, REPLACEMENT OF OR REFUND FOR THE NONCONFORMING PRODUCT(S) AT SELLER’S SOLE OPTION. THERE IS NO OBLIGATION TO REPAIR, REPLACE OR REFUND FOR PRODUCTS
AS THE RESULT OF (I) ACCIDENT, DISASTER OR EVENT OF FORCE MAJEURE, (II) MISUSE, FAULT OR NEGLIGENCE OF OR BY BUYER,
(III) USE OF THE PRODUCTS IN A MANNER FOR WHICH THEY WERE NOT DESIGNED, OR (IV) IMPROPER STORAGE AND HANDLING OF
THE PRODUCTS.
Unless otherwise expressly stated on the Product or in the documentation accompanying the Product, the Product is intended for research only and is not to
be used for any other purpose, including without limitation, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses, or
any type of consumption by or application to humans or animals.
Current product instructions are available at www.thermoscientific.com/pierce. For a faxed copy, call 800-874-3723 or contact your local distributor.
© 2012 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its
subsidiaries. Printed in the USA.
Pierce Biotechnology
PO Box 117
(815) 968-0747
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
5
www.thermoscientific.com/pierce