Application:
Strip Western Blot for for re-probing
and for mass spectrometry
Features:
- Strip blot in 5~15 minutes
- Incubation at room temperature
- No stench smelling without addition of 2-mercaptoethanol or its analogs
- A propriety formulation with an eco-friendly and non-hazardous organic solvent
(with light odor) included to completely disassociate the bound antibodies
Application I. Strip Western Blot for Re-probing
A Comparison of BlotFresh™ Stripping
Reagent with Pierce's Restore™ Blot Stripping Buffer
- Blot Stripped and Re-probed
with the Same Antibody*:
Total Akt (catalog #
A0501-1) with different treatments in NIH 3T3 cells
* Develop 2 minutes with ECL Plus™
and expose 20 seconds on
Kodak negatives
- BlotFresh™
Stripping
Reagent is More Effective in Breaking Down the Antigen-Antibody Complex than Pierce's Restore™ Stripping Buffer:
BlotFresh™ Stripping Reagent* Pierce's Restore™ Stripping Buffer*
Original
Expose
x 1'
Expose
x 5'
Original
Expose
x 1'
Expose
x 5'
* The blots
were initially probed with β-actin antibody with exposure for 10 seconds. Then
the blots were stripped with BlotFresh™ Stripping Reagent for 5 minutes and with
Pierce's Restore™ Stripping Buffer for 15 minutes respectively. After washes,
the stripping effect was tested by development of blots with ECL Plus™ for 2
minutes followed by exposure to Kodak BioMax films for 1 and 5 minutes
respectively
- BlotFresh™
Stripping Reagent Strips
a
Blot followed by Re-probing with a Different Antibody:
A. Blot was probed with
antibody against total Akt (catalog # A0501-1)*.
B.
Blot was stripped with BlotFresh™ Stripping
Reagent and re-probe with antibody against phospho-Akt (catalog # A0503-1)*.
*
Lane 1 is serum starved overnight while lane 2~5 were treated with PDGF in
different concentrations in NIH3T3 cells.
The blots were developed 2 minutes with ECL Plus™
reagent and expose 10 seconds on Kodak negatives (ECL Plus™
is a trademark of GE Healthcare-Amersham)
Application II. Strip
Western Blot for Mass Spectrometry
Procedures for Stripping Western Blot for Mass Spectrometry:
- Strip blot for 15 minutes at RT to
remove primary and secondary antibodies
- Locate the protein by comparing
the stripped blot and exposed film and cut the band from blot
- Digest the cut band followed by
mass spectrometry (see the following picture)
Data Sheet
Testimonials:
...had received sample of BlotFresh™ Western Blot Stripping Reagent. Fantastic
product!! Have used to strip same blot 2x, and resulting three radiographs are
indistinguishable from primary blots with the second two antibodies. I'll be
ordering some today...
------by Dr. Catherine Bair, NCI, NIH
I cannot live without it! It is just amazing. I used to use Pierce’s and I often
need to re-run Western due to dirty blot after strip. Since I started using this
product, my life has been changed… It makes my research easier and I do not need
to re-run gel anymore… And the stripping process is just 2 minutes. It is really
amazing product!
------by Dr. Jennifer Lin, the George Washington University
Some Product Citations:
1. Mahmoud M. El-Mas, Ming Fan, and Abdel A. Abdel-Rahman. Endotoxemia-Mediated
Induction of Cardiac Inducible Nitric-Oxide Synthase Expression Accounts for the
Hypotensive Effect of Ethanol in Female Rats. J. Pharmacol. Exp. Ther.
2008; 324: 368-375
2. Grzegorz
Woszczek, Li-Yuan Chen, Sahrudaya Nagineni, Sara Alsaaty, Anya Harry, Carolea
Logun, Rafal Pawliczak, and James H. Shelhamer. IFN-gamma Induces
Cysteinyl Leukotriene Receptor 2 Expression and Enhances the Responsiveness of
Human Endothelial Cells to Cysteinyl Leukotrienes. The Journal of
Immunology, 2007; 178: 5262-5270
3. David A Baker, Aric Madayag, Lars V Kristianse, James H Meador-Woodruf,
Vahram Haroutunia and Ilangovan Raju.
Contribution of Cystine–Glutamate Antiporters to the Psychotomimetic Effects
of Phencyclidine. Neuropsychopharmacology, 2007; Aug. 29 online
publication
4. Ashley B. Williams and Patricia L. Foster. The Escherichia coli Histone-like
Protein HU Has a Role in Stationary Phase Adaptive Mutation. Genetics.
2007; 177:723–735
