[PM6628] ProMetric™ TriColor Low Range Protein Ladder, 2 to 40 kDa, 250 μl

Yes, 50 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. Typically, pre-stained markers are 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 difficult 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. Additionally, low molecular weight proteins may be affected by variables such as gel type and electrophoresis conditions, which can lead to discrepancies when compared to markers from other brands.

Under normal circumstances, the stripping/deprobing process will have minimal impact on SMOBIO’s Protein Markers/Ladders. However, it’s important to note that the intrinsic properties of small molecules, particularly small target peptides, make them more susceptible to loss during this process. Therefore, it is advisable to avoid stripping whenever possible or to minimize the extent of stripping.

To further reduce any potential effects, avoid excessive washing and refrain from washing at room temperature. Proper control of washes prior to stripping can significantly lessen the impact, but the presence of Tween-20 on the membrane during this process can still adversely affect the performance of SMOBIO’s Protein Markers/Ladders.

For proteins below 30 kDa, a Tricine gel is recommended. It is specifically designed to improve resolution for proteins under 20 kDa, providing better separation and sharper bands for low MW proteins.

 We recommend using a 37.5:1 or 29:1 acrylamide-to-bisacrylamide ratio for optimal performance with PM6628 in Tricine gels

The 19:1 ratio is not recommended due to potential formulation inconsistencies, the possible use of hazardous chemicals, and longer electrophoresis times, particularly when using 18% Tricine gels.

Low MW proteins, typically those below 15 kDa, are prone to "over-transfer," meaning they can be lost during the transfer process. This occurs due to inadequate retention by the membrane and the potential for too-rapid transfer, which can lead to incomplete transfer or loss of the protein from the membrane.

 To optimize transfer for low MW proteins, consider the following strategies:

• Choose appropriate membrane:

Use membranes with smaller pore sizes (e.g., 0.2 μm) to enhance the transfer efficiency of small proteins. Thicker PVDF membranes can improve protein binding and stability. If your target peptides are prone to loss on PVDF, consider using nitrocellulose (NC) membranes, which may provide better retention for low MW proteins.

• Optimize transfer buffer:

For blotting Tricine gels, we recommend using 1× Tris-Glycine Transfer Buffer with 20% methanol. Avoid adding SDS to the transfer buffer, as it can impair protein binding to the membrane. Increasing the methanol concentration in the buffer up to 25% can improve protein binding efficiency. Alternatively, using ethanol instead of methanol can also enhance binding for small proteins.

Rinse the membrane with distilled water immediately after the transfer. Then, proceed with blocking, washing, and incubation at 4°C. If additional immobilization of small peptides on the membrane is required, dry the membrane at room temperature before starting the blocking step. Ensure that Tween-20 is kept at a concentration of 0.1% or less in both the washing and blocking buffers.

If a Tricine gel is run with Tris-Glycine running buffer, the gel will take longer to run and provide poorer resolution, particularly for smaller proteins. This is attributed to a combination of the increased stack area size (as glycine is a slower ion compared to tricine) and the higher ionic strength of the Tricine gel.

 To stain proteins in the Tricine gels with Coomassie dye:

A. Wash the gel with 100 mL of ultrapure water for 1 minute with gentle agitation and discard the wash.

B. Stain the gel with 0.1% Coomassie dye dissolved in 40% ethanol and 10% acetic acid for 20-30 minutes with gentle agitation.

C. Destain the gel in destaining solution Ⅰ (40% ethanol and 10% acetic acid). Incubate with gentle agitation on a rocker for 60 minutes.

D. Discard the solution.

E. Continue destaining with destaining solution Ⅱ  (9.5% ethanol and 7% acetic acid) until bands are very clean. Usually we destain the gel overnight in destaining solution Ⅱ with several Kimwipes present.

F. Transfer the gel to water.