Titration of Blue Prestained Protein Marker, Broad Range (11-250 kDa) #59329 (left). Western blot analysis of #59329 using Anti-Blue (2DF11) Mouse mAb (HRP Conjugate) #46387 (right).
Important: Do Not Boil Protein Marker
1. Thaw the protein ladder on ice.
2. Gently vortex solution to ensure the mixture is homogeneous.
3. Load the appropriate volume of the protein marker per lane, as specified below:
0.75-1.0 mm thick: load 5 μl
1.5 mm thick: load 10 μl
0.75-1.0 mm thick: load 10 μl
1.5 mm thick: load 20 μl
Please note, when using #59329 Blue Prestained Protein Marker, Broad Range (11-250 kDa) with #46387 Anti-Blue (2D2F11) Mouse mAb (HRP Conjugate), it is recommended to load between 0.5 μl-2.5 μl of #59329 to better visualize the separation of proteins while minimizing excess background due to an overabundance of protein.
4. Unused ladder may be returned to -20ºC for long-term storage.
Detection of Protein Ladders:
a) Perform chemiluminescent detection according to the manufacturer's instructions except, at the stage of secondary antibody incubation with the blot, concurrently add the Anti-Blue (2D2F11) Mouse mAb (HRP Conjugate) at a dilution of 1:1000. Incubate for one house at room temperature with gentle agitation.
b) Proceed as usual with the detection procedure.
Blue Prestained Protein Marker, Broad Range (11-250 kDa) is supplied in 62.5 mM Tris-Phosphate (pH 7.5 at -20ºC), 1 mM EDTA, 2% SDS, 10 mM DTT, 1 mM NaN3 and 33% glycerol. Store at –20°C for up to one year.Anti-Blue (2D2F11) Mouse mAb (HRP Conjugate) is supplied in 136 mM NaCl, 2.6 mM KCl, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
The Blue Prestained Protein Marker Detection Pack is designed to detect the molecular weight ladders on western blots when using the horseradish peroxidase (HRP) based western detection system. The pack contains Blue Prestained Protein Marker, Broad Range (11-250 kDa) and Anti-Blue (2D2F11) Mouse mAb (HRP Conjugate). Blue Prestained Protein Marker, Broad Range (11-250 kDa) is a mixture of purified proteins covalently coupled to a blue dye that resolve to a series of 11 bands between 11 and 250 kDa following electrophoresis. The protein concentrations are carefully balanced for even intensity. The covalent coupling of the dye to the proteins affects their electrophoretic behavior in SDS-PAGE gels relative to unstained proteins. The Anti-Blue (2D2F11) Mouse mAb (HRP Conjugate) is conjugated to the carbohydrate groups of HRP via its amine groups. The antibody has been optimized to detect proteins labeled with remazol blue on western blots. The pack is optimized for chemiluminescent western detection procedures.
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