[ DESCRIPTION]
Effective Size Range: 10kDa to 70kDa.
Protein bands: 10 kDa, 14 kDa, 18 kDa, 22 kDa, 26 kDa, 33 kDa, 44 kDa and 70 kDa.
Double intensity bands: The 26 kDa, 18 kDa, 10 kDa bands are at double intensity to make location and size approximation of proteins of interest quick and easy.
Ready-to-use: No need to heat, dilute or add reducing agents before use.
[ STORAGE BUFFER]
62.5mM Tris-H3P04 (pH 7.5 at 25°C), 1 mM EDTA, 2% (w/v) SDS, 100mM DTT, 1 mM NaN3, 0.01 % (w/v) bromo-phenol blue and 33% (v/v) glycerol.
[APPLICATION]
Accurate protein sizing on SDS-polyacrylamide gels and Western blots.
[USAGE]
1. Allow the Marker to reach room temperature and thoroughly mix before use. This will ensure that any solids that may have precipitated at -20°C have returned to solution. Do not boil!
2. Load the following volumes of the Marker on a SDS-polyacrylamide gel.
[ LOADING VOLUMES]
3~5uL per well for mini gel; 7uL per well for large gel.
Use the same volumes for Western blotting applications. The loading volumes listed above are recommended for gels with a thickness of 0.75~1.0mm. The loading volume should be double for 1.5mm thick gels.
[ QUALITY CONTROL]
The protein marker, with molecular weight shifts of < or = 5% and minimal band broadening, are confirmed by migration in SDS-PAGE system. Electrophoresis of the Marker on a 13~18% Tris-glycine SDS-polyacrylamide gel resolves 8 individual bands.
[IMPORTANT NOTES]
1. The protein marker is conveniently packaged and ready to use. There is no need to heat, dilute or add reducing agents. Do not boil, which may cause band degradation.
2. The molecular weights of the proteins have a lot-to-Iot variation of ~5%.
3. These protein markers are for SDS-PAGE and should not to be used for native electrophoresis or a native gel. These markers are denatured and have SDS in the storage buffer.
4. Don't load too much protein. See recommended load volumes in the manual.
5. Check % gel that is being used if the lands miss. Depending on gel type and/or percentage, you may not see all of the bands. For example, one would not see the smallest bands of the standard on a very low % gel. A high % gel may not resolve the higher MW bands. Homogeneous low percentage gels are recommended for analysis of large proteins and high percentage gels for analysis of small proteins. In high percentage gels (13~18%) large proteins (22~70kDa) can separate, while in low percentage gels (4~8%) small proteins (14 and 10kDa) will migrate with the tracking dye. Longer transfer times or higher transfer voltages may be required for Western blotting of large (>100kDa) proteins.
6. If additional bands are observed in the gel image of the protein ladder, this might be caused by DTT oxidation in the storage buffer.
7. If proteins show a poor transfer, increase voltage, current or length of time for transfer, and pay attention to the SDS from the transfer buffer. SDS will cause the proteins to bind less efficiently to membranes because it disrupts the hydrophobic interaction between the membrane and the protein. If SDS is present in transfer buffer, make sure that there is no more than 0.02% in buffer.
8. Alcohol enhances hydrophobic binding to membrane. Not enough alcohol may prevent binding.
[ SHELF LIFE]
Storage: -20 degree C, 1 year at -20 degree C. If the product has been stored as directed for one year, its shelf life maybe extended by adding dithiothreitol (Dn) to approximately 50mM.