To illustrate the suitability of Pall Immunodyne® ABC membrane for reverse dot blot applications.

Using a model system, Immunodyne ABC membrane was dot loaded with amino-terminated oligonucleotides. Following hybridization, the dots were visualized with colorimetric or chemiluminescent detection. Nitrocellulose membrane was used as a control. The results show that higher levels of detection may be obtained on Immunodyne ABC membrane using both detection methods, than nitrocellulose.

This report describes experiments performed to illustrate the suitability of Pall Immunodyne ABC membrane for use in tests which use Reverse Dot Blot technology.   A model system was used with colorimetric and chemiluminescent detection. Nitrocellulose membrane was used as a control.

Membranes Tested
0.45µm Immunodyne ABC membrane, Part No. BC045H5R Lot No.IA0704
Hybond C Nitrocellulose membrane, Part No. RPN 2020W Lot No.1818185

DNA Labelling and Detection Kits
Roche Applied Sciences 3’ tailing kit, Catalog No. 1417231
Roche Applied Sciences DIG colorimetric DNA detection kit, Catalog No. 1175041
Roche Applied Sciences DIG chemiluminescent DNA detection kit, Catalog No. 1363514

Solutions Used
Pre-hybridization solution containing 5 x SSC, 1% (w/v)
Blocking reagent 0.1% (w/v) n-lauryl
Sarcosine, 0.02% (w/v) SDS

Post-hybridisation washes:

1. 2 x SSC, 0.1% (w/v) SDS
2. 0.2 x SSC, 0.1% (w/v) SDS

Detection buffers:

1. 0.1M Maleic Acid 0.15M NaCl pH 7.2 containing 0.3% (w/v) Tween 20
2. Buffer 1 containing 1% (w/v) blocking reagent
3. Tris.HC1 0.1M NaCl 50mM MgC12

Aminoterminated oligonucleotide (21-mer)
Complimentary oligonucleotide

Methods

1. The amino-terminated oligonucleotide was diluted in 6 x SSC to give 1µg, 500, 100, 50, 10, 5 and 1ng/µl.
2. The diluted oligos were dot loaded onto duplicate samples of each membrane.
3. The membranes were allowed to air dry.
4. Unlabelled oligonucleotide was labelled with digoxygenin according to the manufacturer's protocol for the Roche Applied Sciences 3’ end tailing kit.
5. Membranes were incubated in pre-hybridization solution for 1 hour at 68 °C.
6. Labelled oligonucleotide was added to hybridization solution at a concentration of 10pmol/ml.
7. Hybridization was carried out overnight at a temperature of 54 °C.
8. Post-hybridization washes were performed as follows:
   1. 2 x 5 minutes in 2 x SSC, 0.1% (w/v) SDS at 54°C
   2. 2 x 5 minutes in 0.2 x SSC, 0.1% (w/v) SDS at 54°C

Detection

1. Membranes were rinsed for 1 minute in buffer 1.
2. Membranes were blocked by incubation for 30 minutes in buffer 2.
3. Anti-dig-alkaline phosphatase conjugate was diluted 1/5000 in buffer 2. Membranes were incubated in this solution for 30 minutes.
4. Excess conjugate was removed by washing for 2 x 15 minutes in buffer 1.
5. Membranes were equilibrated for 5 minutes in buffer 3.
6. Colorimetric detection was performed using NBT/BCIP, chemiluminescent using substrate (Lumigen, Detroit,, MI, USA) PPD and exposure to x-ray film.

The results show that using the model system outlined the 5ng oligonucleotide can be detected on Immunodyne ABC membrane, compared to 500ng on a nitrocellulose membrane.

This indicates that Pall Immunodyne ABC membrane is a good candidate membrane for Reverse Dot Blot applications using either colorimetric or chemiluminescent detection systems.
  Plate 1. Chemiluminescent Detection of Reverse Dot Blot on Immunodyne ABC and Nitrocellulose Membranes.
Plate 2. Showing Colorimetric Detection of Reverse Dot Blot on Immunodyne ABC and Nitrocellulose Membranes.