The Evolution of Protein Markers
Protein molecular weight markers are indispensable tools in SDS-PAGE and Western blotting, serving as reference standards for estimating protein size and evaluating electrophoresis or transfer performance. Over the years, protein markers have evolved through several key stages — from traditional unstained markers to pre-stained and, more recently, imaging (exposure) markers.
|
Feature |
Unstained Protein Marker |
Pre-stained Protein Marker |
WB Imaging / Exposure Marker |
|
Appearance |
Colorless in solution; bands visible only after staining (e.g., Coomassie or silver stain) |
Colored bands visible during electrophoresis (usually blue, red, or green) |
Chemiluminescent or fluorescent reference bands visible directly on Western blot images |
|
Primary Use |
Accurate molecular weight determination |
Monitoring electrophoresis and transfer efficiency |
Alignment and exposure reference during chemiluminescent or fluorescent WB imaging |
|
Molecular Weight Accuracy |
Highest accuracy (no dye interference) |
Slightly altered migration due to dye conjugation |
Moderate; designed for imaging consistency rather than precise MW |
|
Visualization Before Staining |
Not visible |
Visible during electrophoresis and membrane transfer |
Visible during imaging (after substrate addition or under fluorescence) |
|
Compatibility with Staining |
Compatible with all post-staining methods |
Can be used for visual tracking; some may fade after transfer |
Visible only in imaging step, not for staining or transfer tracking |
|
Quantitative Use |
Suitable for accurate size determination |
For qualitative reference only |
For imaging reference only (signal normalization or exposure check) |
|
Typical Applications |
SDS-PAGE with post-stain detection |
Routine SDS-PAGE and WB transfer monitoring |
Western blot exposure calibration and image alignment |
|
Advantages |
High precision, ideal for molecular weight analysis |
Easy visualization, real-time monitoring |
Direct visibility on WB image, avoids overexposure errors |
|
Limitations |
Invisible before staining |
Slight MW shift due to dye conjugation |
Not suitable for MW estimation or gel monitoring |
Yeasen’s Advanced Pre-stained Protein Markers
Yeasen’s pre-stained markers are based on recombinant E. coli-expressed proteins with uniform sequences, specially designed with His-tags removed and site-specific labeling to ensure consistent dye conjugation. Calibrated against standard unstained molecular weight markers, Yeasen’s Tricolor Marker provides accurate size estimation across a broad molecular weight range—from 2.7 kDa to 300 kDa. The color-coded bands make it easy to monitor electrophoresis in real time and confirm efficient protein transfer to PVDF or NC membranes, where the bands remain clearly visible for downstream analysis.
Features
- Extensive Size Coverage:
Three broad-range markers: 8–180 kDa, 10–180 kDa, and 10–245 kDa
High-molecular-weight marker: 25–300 kDa
Low-molecular-weight marker: 2.7–40 kDa
- Vivid Tri‑Color Staining: Distinct color bands, each at an average concentration of 2.5 µg
- Robust Stability: No degradation at 50 °C 20 h
Applications
- Molecular weight reference for proteins during SDS-PAGE
- Verification of band accuracy in protein expression experiments
- Assessment of protein purification results
- Validation of target protein size during antibody development and processing
Performance Demonstration
Wide Compatibility
Figure 1. Compatible with various gel systems and meets standard requirements for routine electrophoresis.
Visible at Different Loading Volumes
Figure 2. Marker bands are clearly seen in both gel and membrane images. Band intensity increases after transfer.
Brighter Bands
Figure 3. Brighter band colors compared to other markers at the same loading volume.
Thermal Stability
Figure 4. Figure 4. No degradation observed after treatment at 50°C for 20 h.
Enhanced Resolution for Small Proteins
Figure 5. Protein marker during electrophoresis and transfer (left). Yeasen Marker (Cat# 20344) has more bands , making small proteins easier to identify (right).
Frequently Asked Questions (FAQ)
Q1: Why are some bands missing from the protein marker on the gel?
A: Check the type and percentage of polyacrylamide gel used. The gel pore size determines how effectively proteins of different molecular weights are separated.
Typically, a 12.5% gel (acrylamide:bisacrylamide = 29:1) can clearly separate bands between 8–180 kDa. On a 10% gel, the 8, 15, and 17 kDa bands may compress together at the dye front, while on an 8% gel, the 10, 17, and 25 kDa bands may overlap.
Q2: The pre-stained protein marker bands fade or disappear over time—why?
A: This usually occurs due to contamination that introduces proteases or other reactive substances, leading to degradation of the marker proteins.
Solution:
- Use clean, disposable pipette tips and tubes to avoid cross-contamination.
- When the marker is shared by multiple users, aliquot it immediately into clean microtubes after opening.
Q3: The protein marker bands appear smeared or blurry. What should I do?
A:
- Always use ultrapure water when preparing buffers.
- Choose an appropriate gel concentration and make sure the gel is within its effective usage period—old gels can affect resolution.
- Avoid excessive voltage or extended running times that may cause overheating.
- Replace old or improperly buffered electrophoresis buffers and pre-chill them before use.
- Prevent contamination during storage or sampling by using clean pipette tips and tubes.
Q4: Can pre-stained protein markers be used in native (non-denaturing) PAGE?
A: It is not recommended. Pre-stained markers are already denatured and partially reduced in SDS buffer. Their indicated molecular weights do not reflect native protein mobility. However, they can still serve as a rough migration reference during native electrophoresis.
Q5: Why do Yeasen’s pre-stained markers show slightly different apparent molecular weights compared to other brands or to my target protein?
- A: Apparent molecular weight can vary among brands and buffer systems due to:
- Different calibration standards used for validation (non-prestained markers).
- Variations in amino acid composition and SDS binding efficiency among proteins of the same nominal molecular weight.
- Distinct dye-labeling chemistries, which can alter protein migration behavior under different buffer systems.
Related Products
|
Category |
Name |
Cat. No. |
Size |
|
Wide Range Protein Marker |
GoldBand™ Plus 3-color Regular Range Protein Marker(8-180 kDa) |
20350ES72/76/90 |
250 μL/2×250 μL/10×250 μL |
|
GoldBand™ 3-color Regular Range Protein Marker(10-180 kDa) |
20351ES72/76 |
||
|
20352ES76/90 |
2×250 μL/10×250 μL |
||
|
Low Molecular Weight Protein Marker |
20344ES72/76/90 |
250 μL/2×250 μL/10×250 μL |
|
|
High Molecular Weight Protein Marker |
GoldBand™ Plus 3-color High Range Protein Marker(25-300 KDa) |
20347ES72/76/90 |
|
|
Unstained Protein Marker |
Unstained Protein Ladder（10-200 kDa） |
20312ES76 |
2×250 μL |
|
Unstained Protein Ladder（14.4-116.0 kDa） |
20313ES76 |