A. Samples for EMSA
Samples for EMSA require that nucleic acid-binding proteins are 1) non-denatured, native, 2) activated, 3) at a levels enabled detected by chemilumincence or fluorescence. Some kits of nuclear protein extraction are designed for detection of protein-translocation, not suitable for EMSA. Some kits of nuclear protein extraction are easy to cause contamination of extracts with cytoplasmic proteins, which may reduce the percentage of protein factors in the extract, decrease the signaling of DNA/RNA protein complexes, cause high background. Viagene Biotech provides nuclear extraction kits specifically for EMSA (check).
B. Types of samples for EMSA
One advantage of EMSA is detection of specific protein factors from whole cell lysates, nuclear extracts. Of course, purified or partial purified proteins can also be used for EMSA. Furthermore, the proteins form gene express in cell-free translation system can be used for EMSA if the proteins have activity of binding DNA/RNA.
C. Problems with samples for EMSA
1. No DNA/RNA binding proteins.
Before sample preparation, Google to check if the cells have target protein express, otherwise, expression vectors should be transfected into cells and level of expressed proteins should be check by Western blot or immuno-fluorescence.
2. DNA/RNA binding proteins is inactivated
Many protein factors need to be activated for binding to nucleic acids. Inactivated proteins are from the following factors: (1) cells does not undergo any activations and proteins is inactivated. 2) Most activated protein factors can only last several hours. Protein factors are returned to inactivated status if cells are not collected in time windows. 3) Improper treatment of cells with activated chemicals or drugs; for the activating factor binding to cell surface, one-hour treatment is good enough to activate protein factors, for the drugs or chemicals function inside cells, ~6 hours treatment is good enough to activate most of protein factors. 4) inactivated protein can still be observed from cells without specific cell receptor(s) binding to a stimulating chemicals or factors even the procedure of activation is carried out correctly.
Protein factors become activated after undergoing series of response such as biochemistry change, phosphorylation, protein degradation, IR multimer formation, or nuclear translocation etc. The activation of protein factors varies from cell lines to other one, from one protein factor to another, which can be determinate by pre-experiments or from published articles. For our customer using our EMSA service, we willing to help our customers to increase the successful EMSA.
3. Proteins in sample are degraded
After cell treatment, the flaskets/dishes should keep on ice and protein extraction is carried out at low temperature. All the buffers or solutions are pre-cold. using protease inhibitors to prevent degradation. The extracts should be kept in a ultra-low freezer or a liquid- nitrogen tank.
4. Purified protein loses its nucleic acid-binding activity
Purified protein factors may lost their activity of DNA/RNA binding if a protein is express in bacteria. Purification may change the structure of purified protein, separate protein from other submembers.
5. Protein is inactivated when expression in bacteria.
Some protein factors require translational modification, such as sugar chain or -s- chains, for their functions. When those protein are expressed in bacteria, they lost their activity due to that the translational modifications cannot be carried out in bacteria or incorrectly.
Viagene Biotech provides a variety of EMSA services (detail). The features of our service are 1) we have extensive experience, serviced hundreds of customers. 2) we have facility to carry out any EMSA or EMSA-related services. 3) Fast results, usually 7-10 working days. 4) we have variety of EMSA controls and reagents to help us to solve EMSA problems.