EMSA/Kit Choice

  
        

Performing EMSA service many years, our scientists are very familiar with EMSA products on the market. The follows are comments for EMSA kits on the market.

A. Principle of choosing EMSA kits:

(1) How many years are the kits on the market and how many have customers used them?
(2) Does the company provide EMSA-related products?
(3) Contact the companies selling the kits. If the customer supports know less about EMSA than you, your problems with EMSA, most likely, cannot be solved if you run into trouble with EMSA.
(4) Are the customers satisfied with their purchasing?
(5) There are publications supporting the kit at working. Although no publication does a indicator of non-working kits, but publications definitely are positive indicators for good products
(6) Does not use several products from different companies because 1) components of kits from different sources are not same, mixing use of which may not work, 2) it would be difficulty to figure out the problems.

B. Pro and Con of EMSA kits on the market

Viagene understands EMSA technology and EMSA products on the market very well. The follows list a variety of EMSA kits on the market as a guidance for customer to choose a proper EMSA products.

1. Roche DIG EMSA kits:

Pro -- Non-radioactive EMSA

Con -- Before Biotin- or IR-probes are used for EMSA, DIG EMSA is a only non-radioactive EMSA on the market. However, it does not have any advantage than EMSA using biotin/IR-probes and should be avoided for these reasons; 1) Price is higher than other EMSA kits. 2) Manual labeling; when DIG kit is used, probe-labeling can only be carried out manually at lab, which will take at least 2 days to get probes and the efficiency of labeling is un-constant. However, biotin/fluorescence can be used to label nucleic acids during DNA synthesis, which is as simple as order an oligo. With Viagene's custom probe service, customers can get a probes ready for EMSA. 3) Time-consuming,, DIG kit is based on antigen/antibody detection. The interaction of ag/ab would be twice, taking at least 1 hour for each incubation. Taking ~8 hours to get the images. However, biotin/avidin interaction is 10,000 fold higher than that of ag/ab interaction, and can be completed ~10 minutes. Taking 5 hours to get the results. When IR Fluo--probes are used, all steps are omitted and the result can be obtained ~2 hours. 4) High background may be observed when DIG kit is used because ag/ab system can only tolerated to gent incubation and washing. However, biotin/ECL EMSA can undergo through more strict buffer incubation with much low background than that of DIG system. 5) Lower sensitivity of DIG system using alkaline phosphatase is than that of HRP/ECL of biotin/avidin system. HRP/CEL system is much popular than that of AP chemilumincence system. If digital imager is used, AP/chemilumincence may not be detectable.

2. Invitrogen gel-staining EMSA kit:

Pro -- Non-radioactive EMSA, simple operation and low costs.

Con -- 1) low sensitivity when ologos are stained with SYBR green. 2) amounts of probes and nucleic acid-binding proteins are used much higher than that of EMSA using labeled probes. 3) As poly[dI:dC] can be stained with SYBR green as well, poly[dI:dC] can not used with gel-staining EMSA, which would cause non-specific background and is not suitable for cell or nuclear extraction, but good for purified proteins. 5) Ruby-staining of purified proteins is unnecessary as purified proteins, usually, should be identified during or after protein purification.

3. Thermo/Pierce Light Shift EMSA kits:

Pro -- (1) Non-radioactive detection. (2) HRP/ECL system with high sensitivity. (3) long lasting. (4) with EBNF control (but, it is not a good one for following reasons):  .

 (1) EBNF band locates at the position just above free probes, much smaller that that of most nucleic acid-protein complexes. It is too small to locate most of target proteins.
((2) Probes & samples of EBNF control are completely different from that customer want to use, therefore,  the control cannot provide information for the real reaction of target probes & samples. If specific shifted bands cannot be observed, the problems could be 1) low level of nuclear extracts, 2) proteins degradation, 3) inactivated protein factors or 4) non-working probes etc, no of which has relationship with EBNF controls.
(3) EBNF control was considered to be an indicator for working EMSA as EBNF band cannot be seen if electrophoresis, trans-blotting, UV crosslinking, biotin/avidin reaction, reagents or non-working ECL substrates etc. do/does not work. However, if problems mentioned above happen, free probe bands could not be seen either. Since free probes can be used as indicators, why needs EBNF control.
(4) EMSA pictures with free probe lane(s) can be used for publication directly, however, it would be strange if EMSA pictures include EBNF band(s).

Instead, Viagene Biotech provides real controls of common protein factors, such as NFkB/NK-kB, AP1, SP1, OCT1, OCT2, NRF2, GATA, STATs, WT-1, E2F, P53...(details).

Con -- (1) price is high. (2) no any probe is included. Customers need to get their own biotin- labeled probes, which may not work if a probe sequence is copied from a published article. (3) The users need to perform experiments to confirm the interaction of nucleic acid and proteins. According to the manufacturer, the components of kits could cause big deference for detecting a same factor (check to see). (4) no trans-membrane is included, many membranes in user's lab may not work at all.

C. Li Cor IRDye EMSA kits:

Pro -- 1) Non-radioactive detection. 2) IRDye-probes with high sensitivity. 3) Provide general kit and several ready probes.

Con -- 1) Price is high $60/25 assays (Viagene Biotech $115/50 assays or $199/100 assays). 2) only a fow ready probes available (Viagene provide >100 ready probes). 3) Aggregation seems noticeable. 4) no custom probe service (Viagene's custom probe services).

D. Signosis & Panomics EMSA kits:

Pro -- 1) Non-radioactive detection. 2) HRP/ECL system with high sensitivity. 3) EMSA kits with probes.

Con -- 1) Price is higher than that of Viagene's. Panomics $425/25 assays, Signosis $359/30 assays (Viagene Biotech $279/30 assays or $379/50 assays. 2) No IR Fluo-EMSA kits available (Viagene provides 100+ IR EMSA kits).

E. Kits for nuclear protein extraction:

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 plasmic 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).

F. Kits for determination of protein concentration:

Protein concentration of nuclear extraction is, usually, at the range of 1-3 ug/ul and 15-30 ug/ul for samples for Western blotting. Therefore, protein concentration for EMSA need to be measured accurately; 1) BCA kits, but not Bradford, Lowry or Folin-phenol, are recommended for EMSA for accurate results. 2) Nuclear extraction kits may contain EGTA, DTT etc. and cause false high protein concentration. 3) A low range (0-8 or 10 ug/ul) of protein standard curve is required for the determination of nuclear protein concentration. Viagene provides a kit for detection of low level of nuclear protein concentration (check).

 

 

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