Protein Marker Related Questions
ExcelBand™ Protein Marker
Why are there contrasting results in molecular weights after using different brands of protein markers?
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.
Protein marker Retention Period: Mentioned -20°C and over 2 years. Is it available for 30 months or 36 months? Have you tested this period?
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.
How many times of freezing and thawing are available for protein markers? If it uses 5 μL per load, would the total usage quantity be 50 times x 2 (250 μL x 2 tube)?
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.
Do you have data comparison for protein molecular weight’s precision with other protein markers?
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.
Will SMOBIO’s Protein Markers/Ladder be washed out during Western blotting process?
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.
Will SMOBIO’s Protein Markers/Ladder be affected by the stripping/deprobing process with the presence of β-Mercaptoethanol (β-ME)?
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
Why the upper bands of the protein markers are missing on NC/ PVDF membrane after transferring?
Long transfer time or high transfer voltage may be required for transferring of large protein (> 100 kDa)
Why the bands of protein markers are faint on gel?
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.
The upper bands of the protein marker are missing. What could be the reason?
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.
How to avoid contamination of protein marker by protease?
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.
YesBlot™ Western Marker I (WM1000)
What is the composition of the YesBlot™ Western Marker I?
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.
Does YesBlot™ Western Marker I selectively bind to IgG antibodies generated from different host species?
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.
Can YesBlot™ Western Marker I be used for protein quantification?
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.
Will the YesBlot™ Western Marker I be detected using IgM antibodies?
YesBlot™ Western Marker I contains IgG binding proteins, which will not bind to IgM antibodies.
Can YesBlot™ Western Marker I be used with fluorescent-dye conjugated secondary antibody?
YesBlot™ Western Marker I can bind to IgG antibodies labeled with fluorescent dye.
I used WM1000 and did not see some of bands after western detection. What could have happened?
Here are possible causes and solutions
A. Not enough volume loaded: Load more western markers.
B. Not enough antibody incubation: extend the incubation time for 2hr at room temperature.
C. Not suitable primary antibody diluent: try to use 2% BSA as diluent or follow the instruction from antibody supplier.
D. Not suitable transfer membrane: try to use 0.22 μm PVDF
I used WM1000 and did not see high molecular weight bands (>100 kDa) after western detection. What could have happened?
Incomplete or poor transfer: Longer transfer time or high transfer voltage may be required for western blotting of high molecular weight bands ( > 100 kDa).
Why the signal of low molecular weight bands is faint after western detection?
Here are possible causes and solutions
A. May be masked by the prestained protein: extend the electrophoresis time to separate prestained protein from the low molecular weight bands.
B. Not suitable transfer membrane: try to use 0.22 μm PVDF.
C. Inappropriate methanol concentration:
(a) Use freshly prepared transfer buffer.
(b) Try to increasing the methanol concentration in the transfer buffer (up to 15% ~ 20% v/v methanol).