Western Marker Neo (wide)
Cat. No.: FNK-BCL-WMN-22
Size: 250μl
Storage: -20℃
Description:
Western Marker Neo is the new generation marker protein for western blot with IgG-binding proteins and blue-stained proteins. With the marker one can visually check the transfer of proteins to membrane, and detect the marker protein by enzyme-labeled antibodies by ECL substrates. Thus, Western Marker Neo is very useful for many researchers. The marker is ready to use for SDS-PAGE.
Principle:
Western Marker Neo is composed of 11 recombinant IgG-binding proteins. Ten IgG-binding proteins having molecular weight between 20 to 180kDa are all tightly bound to IgGs of most animal species. Membrane-transfered maker proteins along with target proteins after separation by SDS-PAGE, react with primary and enzyme-labeled secondary antibodies, and shows chemiluminescence after substrate addtion.
The product also includes five blue-colored protein of molecular weight of 108, 69.3, 49.8, 32.2, 16kDa. The dye is covalently bound to the proteins so that one can easily chck the separation of proteins during SDS-PAGE and transfer efficiency. The colored proteins do not interact with IgG-binding proteins.
The apply volume is dependent on the antibody (species of origin and concentration), but the recommended applied volume is between 1 and 5 µL. Too much application can cause too strong of a signal, as well as the appearance of contaminated or degraded protein bands.
Features:
1. Transfer efficiecy and molecular size confirmable
2. Selectable from broad molecular weight proteins
3. Ready-to-use
4. High cost performance
How to Use
＜General instructions＞
- The application volume to gel is 2～5 μl/well.
- Apply directly to gels, no-pre-treatment is needed.
＜Detailed instructions＞
1. Apply to SDS-PAGE gel.
Apply 2～5 μl of Western Marker Neo to gel wells along with your pre-treated sample, and separate proteins by electrophoresis.
【note 1】Change the apply volume depending on your experimental condition.
【note 2】During electrophoresis, blue line may be appear on the front.
2. Blotting to membranes
Take out the gel, set on the blotting apparatus, and transfer proteins to the membrane. Confirm that the blue-colored proteins can be seen in marker lane on the transferred membrane (refer to the figure in page 2)
【note 3】In some occasions blue color may be blotted on the front part in the transferred membrane, but the color will disappear in the following processes, and does not affect antibody reaction.
3. Blocking
Treat membranes with appropriate blocking solution.
4. Primary antibody reaction
Treat membrane with appropriate primary antibody solution. Primary antibody also binds to marker proteins during the reaction.
5. Secondary antibody reaction
Treat membrane with appropriate enzyme-labeled secondary antibody solution.
Secondary antibody also binds to marker proteins directly or via the bound primary antibody during the reaction.
【note 4】As the labeled enzymes on secondary antibody, HRP (Horse Radish Peroxidase) or AP (Alkaline Phosphatase) can be used.
Identification of marker proteins
Signal intensities of marker proteins are designed to be different each other. For example, 21 kDa protein shows a strong signal, 30 kDa protein shows a weak signal, and 190 kDa protein shows a strong signal. The bands of 65 and 72 kDa proteins are in close proximity. These are good marks for identifying MW.
【note 6】Higher antibody concentration, which intensify marker signal, may cause additional band (refer to (3) Product Information for detail).
【note 7】The signal strength of marker is dependent on the origin (animal species) of antibody.
Troubleshooting
| Problem |
Cause and Solution |
| Marker signal is too weak |
1. Marker volume not enough. Increase volume. |
| 2. Low antibody concentration. Increase concentration. |
| 3. Transfer not enough. Increase transfer current or time. |
| 4. Too much transfer time and current. With nitrocellulose membrane, proteins go through the membrane. Decrease transfer current or time, or change membrane to PVDF. |
| Marker signal is too weak |
5. Marker volume too much. Decrease volume. |
| 6. High antibody concentration. Decrease concentration. |
| 7. Too long incubation time. Shorten the incubation time. |
| Fall out a part of band signal |
8. In ECL, strong signal is suppressed. Decrease the marker volume, or reaction time. |
| High background |
9. High antibody concentration or long incubation. Decrease concentration or incubation time. |
| 10. Not enough blocking. Increase the blocking time or other conditions to increase blocking, or change the species of blocking agent. |
| 11. Not enough Washing. Increase the number or time of washing. |
