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Strip Western Blot for for re-probing and for mass spectrometry
- 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)
...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
Note: All prices in US Dollars