Thermo Scientific Pierce Reversible Protein Stain Kits for Membranes are rapid and sensitive alternatives to Ponceau S stain for protein detection on nitrocellulose or PVDF membrane after transfer from polyacrylamide gels.
These kits for membrane-staining use a nondestructive, reversible, reliable and sensitive method to stain and detect proteins on nitrocellulose and PVDF membranes. These Pierce Kits are superior alternatives to Ponceau S stains for evaluating the efficiency of protein transfer following SDS-PAGE and before immunoblotting. The lower limit of detection with this method is 25 to 50ng per band (at least five times more sensitive than traditional Ponceau S staining). The staining protocols are simple, quick and result in turquoise-blue bands that do not fade and are easily photographed for future reference. The stain can be easily reversed in less than 15 minutes. Subsequent Western blot detection is unaffected because the stain does not alter the protein and is completely removed. Separate kits for nitrocellulose and PVDF membranes use the same dye-binding mechanism but differ in the stain formulation and membrane-treatment components to optimize the speed and performance with each type of membrane.
- Better than Ponceau S – more sensitive, easier to document, permanent until reversed (does not fade)
- Sensitive – high-avidity, total-protein stain; lower limit of detection equals 25 to 50ng per band
- Specific – detects only protein; does not bind or interact with other electrophoresis or sample components
- Rapid – stain in less than 5 minutes (nitrocellulose kit) or 30 minutes (PVDF kit); completely erase and reverse staining in less than 15 minutes
- Stable – components are stable at room temperature, saving refrigerator space and eliminating equilibration steps
||Thermo Scientific Pierce Reversible Protein Stain and Ponceau S stain. A comparison of GST lysate staining on nitrocellulose. Increasing amounts of GST Lysate protein were applied onto two 4-20% Tris-glycine SDS polyacrylamide gels. Proteins from both gels were electroblotted to nitrocellulose membrane. Panel A. was treated with Pierce Reversible Stain for 30 seconds and destained according to the protocol. Panel B. was stained with 0.1% Ponceau S stain for 5 minutes and destained. The blot stained with Pierce Reversible Stain demonstrates superior visual detection of bands. GST Lysate loading volumes (Lane 2-4). Lane 1. Prestained MW marker (Part No. 26681), 10µL Lane 2-4. 15µL, 10µL and 5µL lysate.
Specifications compared to Ponceau S reversible stain.
|Thermo Scientific Pierce
Reversible Protein Stain
|Tight-binding, high-sensitivity total protein stain
||Weak-binding, low-sensitivity total protein stain
|Detection limit: 25-50ng
||Detection limit: 250ng
|Turquoise blue bands easily photographed
||Low-contrast red bands difficult to photograph
|Bands do not fade but can be erased
||Stained bands fade within hours
|Typical staining time: 1 minute
||Typical staining time: 5 minutes
|Background eliminated quickly with low pH wash
The Pierce Reversible Protein Stain Kits for Nitrocellulose and PVDF Membranes have excellent avidity for a broad range of proteins, resulting in low protein-to-protein variability and enabling most proteins to be detected at very low levels (25 to 50ng per band). These Pierce Membrane Stains are superior to other protein stains available for nitrocellulose and PVDF membranes. For example, Ponceau S is less sensitive and results in red bands that easily fade and are difficult to photograph. Coomassie dye is a sensitive stain, but it permanently binds to proteins and can interfere with Western blotting.
Pierce Reversible Protein Stain Kits for Membranes use a dye that has a high affinity for protein but does not permanently bind. The stain has minimal nonspecific interactions with the membrane surface and protein-transfer reagents, and the technique is compatible with general Western blot systems. The treated membrane does not interfere with conventional chemiluminescent or chromogenic detection using HRP and alkaline phosphatase substrates. In addition, the stains are compatible with N-terminal sequence analysis of proteins excised and eluted from membrane.
Because of the different properties of nitrocellulose and PVDF membranes and their interaction with Pierce Reversible Protein Stain, two kits have been developed which are optimized for staining proteins on either nitrocellulose or PVDF membranes. It is important to note that these two kits, while they contain similar reagents, are not interchangeable for detecting proteins on nitrocellulose and PDVF membranes.
|Comparison of Thermo Scientific Pierce Reversible Protein Stain with Ponceau S stain. Unstained Protein MW Markers were serially diluted and applied to two 4-20% Tris-glycine-SDS polyacrylamide gels (Lanes 2-10). Proteins from both gels were electroblotted to PVDF membrane. Panel A. Blot stained with Pierce Reversible Stain for 1 minute and destained according to the protocol. Panel B. Blot stained with 0.1% Ponceau S in 5% acetic acid for 5 minutes and destained according to the published protocol. Lane 1. Prestained MW marker (Part No. 26681).
||Enhanced Western blotting following staining with Thermo Scientific Pierce Membrane Stain. Different amounts of purified GST protein were applied to two 10% Tris-glycine SDS-polyacrylamide gels. Proteins from both gels were electroblotted to nitrocellulose membranes.
Panel A. Western blot was not treated with a reversible stain.
Panel B. Western blot was subjected to the staining, destaining and stain erasing protocol of the Pierce Reversible Stain Kit. Both membranes were then probed with anti-GST incubated with goat anti-rabbit IgG-HRP conjugate (Part No. 31460) and detected using Thermo Scientific SuperSignal West Dura Substrate (Part No. 34075).
Lanes 1 to 4: 125pg, 250pg, 500pg and 1ng GST protein.
Membranes for Western blotting
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