[PM2700] ExcelBand™ 3-color Broad Range Protein Marker (3.5-245 kDa), 250 μl x 2

A. Different proteins even with similar molecular weights would exhibit apparent disparity from the resulting SDS PAGE due to the difference in the composition of the protein’s amino acids (e.g. gelatin). The reason for the disparity is due to the amino acids composition that affects the binding of the protein and SDS. Therefore, we can say that protein marker is a handy tool to estimate molecular weight, but there is no absolute molecular weight standard. 

B. While running SDS-PAGE, protein mobility can be affected by the composition of the buffer used, gel percentage, the voltage used, running time, as well as if there is a pre-run. 

C. Another recommendation for high molecular weight proteins is to prolong the running time to clarify the relative location of bands.

Yes, we have tested our PM2700. The results showed that the PM2700 is stable at -20℃ for at least two years. It has also shown strong performance for more than 36 months under our careful storage. However, we must only suggest a 2 year retention period for the following reasons: There may be a variation in the environment in storage, and improper use may lead to accumulated damage to the proteins and therefore reduce its retention period. 

Yes, 100 uses (5 μL each time) can be expected if freezing and thawing are conducted carefully and properly at the appropriate temperature. Before each use, make sure the protein marker is thoroughly thawed. 

Yes. Usually, pre-stained marker is written on “estimated molecular weight” for caution. It is known that the analysis of protein size by an SDS-PAGE is only for “estimation” because of the intrinsic variation of amino acid composition in all proteins including stained and non-stained ones. For example, a protein which is highly hydrophilic might show a particular higher position in the SDS-PAGE analysis when compared to a hydrophobic one. We did compare the migration patterns of SMOBIO’s Protein Markers with other brands, and we concluded that it was difficult to define “precision” due to the reasons mentioned above. Therefore, in the product description, we suggest our users to calibrate the MW against their interested proteins. Although it is impossible to define "precision" for molecular weight of proteins in SDS-PAGE, we did compare the migration pattern of pre-stained markers with unstained protein marker (Invitrogen MARK12) for calibration. It is concluded that the estimated molecular weight of SMOBIO’s pre-stained marker shows a curve matching well with that of unstained native proteins (MARK12), representing a good estimation of the MW of each pre-stained protein in the SDS-PAGE analysis.

SMOBIO’s Protein Markers/Ladder will be only slightly washed out during Western blotting process. However, the excess of Tween-20 (more than 0.2%) in washing buffer will affect SMOBIO’s Protein Markers/Ladder on the transfer membrane.

Here are suggestions for Western blotting process:
1. Transfer SMOBIO’s Protein Markers/Ladder to membrane with transfer buffer containing 20% methanol to fix SMOBIO’s Protein Markers/Ladder on membrane. 
2. Wash membrane with PBS or TBS containing less than 0.1% Tween-20. 

In normal circumstances, the presence of βME during the stripping/deprobing process will only slightly affect SMOBIO’s Protein Markers/Ladder. However, the presence of Tween-20 on PVDF membrane during the stripping/deprobing process has adverse effects on SMOBIO’s Protein Markers/Ladder.

Here are suggestions for Western stripping/deprobing process:

In the SDS-PAGE, proteins are stacked in the stacking gel and migrate between a highly mobile leading ion (in the gel buffer) and the slower trailing ion (in the running buffer).

These stacked protein bands undergo sieving once they reach the separating gel. However, the resolution of smaller proteins (<10 kDa) is hindered by the continuous accumulation of free dodecylsulfate (DS) ions (from the SDS-containing sample and running buffers) in the stacking gel. This zone of stacked DS micelles causes mixing of the DS ions with the smaller proteins resulting in blurry bands and decreased resolution.

Therefore, it is challenging to detect low molecular weight proteins (<10 kDa) due to insufficient stacking in the gel. In order to avoid insufficient stacking and to increase sharpness of small proteins e.g., 5 kDa of protein, we provide suggestions as the following link: https://www.smobio.com/FAQ-protein-marker