EMSA gel-to-nylon transfer with a fast semi-dry blotter

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Transfer EMSA gels using the Pierce G2 Fast Blotter

The Pierce G2 Fast Blotter provides rapid semi-dry transfer of DNA (TBE) gels to nylon membrane.

Nicholas G. Theodorakis, Ph.D.;

May 22, 2014


The Pierce G2 Fast Blotter (Part No. 62288) is a device designed primarily for semi-dry transfer of protein gels for Western blotting. We were interested to know if the same device would work effectively to transfer DNA-based polyacrylamide gels for performing membrane-based chemiluminescent electrophoretic mobility shift assays (EMSA). We compared the Pierce G2 Fast Blotter and a conventional wet tank method for transfer efficiency of a DNA gel (TBE) to nylon membrane and subsequent performance in a a gel-shift assay using the LightShift Chemiluminescent EMSA Kit (Part No. 20148).


RESULTS and DISCUSSION:

Both the Pierce G2 Fast Blotter semi-dry transfer system and the traditional wet tank transfer were able to transfer EMSA gels to nylon membranes for subsequent detection by chemiluminescence (Figure 1). In this experiment, the G2 Fast Blotter performed better than a wet tank transfer. Although the wet transfer can likely be improved by using a longer transfer time, the G2 Fast Blotter was able to perform the transfer faster, easier, and with less buffer.

Figure 1. EMSA gels transferred using the Pierce G2 Fast Blotter or a wet tank transfer system.

Figure 1. EMSA gels transferred using the Pierce G2 Fast Blotter or a wet tank transfer system. Control EBNA binding reactions from the LightShift Chemiluminescent EMSA Kit were performed and run in duplicate on a 6% polyacrylamide gel in 0.5X TBE buffer. Gels were transferred to nylon membrane using either the Pierce G2 Fast Blotter (lanes 1-3) or a wet tank transfer system (lanes 4-6). Biotinylated probes were detected using the Chemiluminescent Nucleic Acid Detection Module (part of the LightShift Kit) and imaged using the myECL Imager. The open triangle indicates the position of the DNA target duplex (60bp), and the filled triangle indicates the position of the DNA:protein complex.

These data demonstrate the utility of the Pierce G2 Fast Blotter in transferring other types of gels in addition to protein gels for which the instrument was designed.


METHODS:

A complete set of three control EBNA binding reactions from the LightShift Chemiluminescent EMSA Kit (Part No. 20148) were performed according to the product instructions:

  1. Biotinylated DNA target
  2. Biotinylated DNA target + EBNA extract
  3. Biotinylated DNA target + EBNA extract + unlabeled DNA target (competitor)

Forty microliter binding reactions were performed, and samples were loaded in duplicate on a 6% polyacrylamide gel in 0.5X TBE buffer. The gel was electrophoresed at 100V for 60 minutes, and then cut in half. One half was transferred to positively charged nylon membrane (Part No. 77016) with a standard wet transfer in 0.5X TBE at 180mA constant current (about 70V) for 30 minutes. The other half was transferred using the Pierce G2 Fast Blotter in 0.5X TBE at 25V for 15 minutes. The blots were crosslinked using the auto setting of a UV lamp device, then probed with streptavidin-HRP conjugate and detected with chemiluminescent substrate according to the LightShift Kit instructions. Finally, blots were imaged using the myECL Imager (Part No. 62236).