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:

1. Wash the PVDF membrane in methanol for 5~10 minutes prior to the stripping/deprobing process to mitigate the adverse effect of Tween-20.
2. Recommended stripping buffer (for 1 L):  
    15 g glycine, 
    1 g SDS, 
    10 mL Tween 20. 
    Dissolve in 800 mL distilled water. 
    Adjust pH to 2.2
    Bring volume up to 1 L with distilled water 

Long transfer time or high transfer voltage may be required for transferring of large protein (> 100 kDa)

It may be caused by inappropriate electrophoresis condition caused either by SDS-PAGE gel or electrophoresis buffer.

Here are suggestions: 

A. Confirm the APS and TEMED is fresh and prepare the electrophoresis gel freshly.

B. Use fresh electrophoresis buffer in both the inner and outer tanks and avoid excessive reuse of the electrophoresis buffer. 

C. Use a new tip for each sampling to avoid proteinase contamination.

1. The high molecular weight proteins of protein markers are prone to be degraded by proteases. Proteases are found in animals, plants, bacteria, archaea, and viruses, therefore, a general recommendation would be to avoid conducting SDS-PAGE in the same room where the prepared samples are obtained from those organisms.

2. Protein marker is probably contaminated with proteinase K, which is a broad-spectrum, nonspecific, proteolytic enzyme widely used to eliminate protein contaminants in nucleic acid preparations.

1. We would recommend preparing fresh buffers, cleaning the equipment, and using clean pipettes and autoclaved tips when performing the SDS-PAGE.

2. Use new pipette tips before loading each time to avoid contamination. If your tip touches a sample, make sure not to put the tip back into the protein marker tube.

3. Once the protein marker is contaminated, please use a new tube of the protein marker.

4. To avoid cross-contamination of the stock protein marker, it is recommended to aliquot the protein marker into small tubes before use.

1. Gel with 15% acrylamide in Tris-Glycine system is usually able to well separate small proteins ranging from 5 to 10 kDa.

2. Gel with higher than 15% acrylamide can be used, however, less satisfied resolution of protein separation is frequently observed due to technical difficulty in preparation of gel higher than 15%, including uneven polymerization, bubble formation, and most importantly, the ambiguous results for the small proteins/ peptides. If gel with higher than 15% acrylamide is needed to be used, we suggest:

a. Minimize the length of stacking gel. The long length of stacking gel will lead to insufficient stacking of very small proteins, and thus causing ambiguous result. Less than 5 mm (from the well bottom to the top of separation layer) of stacking layer is suggested.

b. Adjust voltage and time of electrophoresis. To obtain better stacking of small   proteins, 100V/15min followed by 150V/ 60min is worthy of try when running with a 20% TG gel.

c. Use loading tip.  When loading the protein marker or the protein samples, we suggest using loading tip which can easily get close to the well bottom and therefore minimize the problem of insufficient stacking of very small proteins.

3. Try using a Tricine gel. Tricine gels are ideal for separation of peptides and small proteins with a molecular weight <10 kDa. Superior resolution is achieved by slowing the migration rate of the peptide-SDS complexes. This helps achieve separation from the faster-moving SDS micelles that interfere with peptide   resolution in Tris-glycine buffer systems.