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. 

YesBlot™ Western Marker I contains ten IgG-binding proteins that binds the primary or secondary IgG antibody used to detect target protein, allowing you to detect the YesBlot™ Western Marker I directly. Addition to IgG-binding proteins, YesBlot™ Western Marker I contains 4 pre-stained proteins (10, 25, 45 and 70 kDa) for monitoring protein separation during SDS-PAGE, verification of Western transfer efficiency on membranes (nitrocellulose, PVDF, or nylon) and for approximating the protein size.

YesBlot™ Western Marker I can bind to IgG antibodies generated from mammalian host species, such as Human, Horse, Cow, Pig, Rat, Mouse, Rabbit, Goat, Sheep, Hamster, Guinea Pig, and etc.

Since Western blot is a series of signal amplification, different kinds of primary antibodies and secondary antibodies will lead to different signal intensity on the film. We do not recommend using YesBlot™ Western Marker I for quantification purpose.

YesBlot™ Western Marker I contains IgG binding proteins, which will not bind to IgM antibodies.

YesBlot™ Western Marker I can bind to IgG antibodies labeled with fluorescent dye.