Blocking Solution for D3308

Item Number

B752099

• Stability And Storage: Store all reagents in the kit at -20℃.
• Storage Temp: Store at 2-8°C
• Shipped In: Wet ice

Product Description

Aladdin's Chemiluminescent Biotin-labeled Nucleic Acid Detection Kit employs Streptavidin-HRP and ECL Moon reagent for chemiluminescent detection of Biotin-labeled nucleic acid. It is suitable for the detection of biotin-labeled DNA or RNA probes in experiments such as Southern blot, Northern blot, ribonuclease protection assay (RPA) or EMSA. This kit is not suitable for the detection of biotin-labeled proteins.This kit also provides the necessary reagents for detection such as blocking solution and wash buffer.This kit uses high-quality Streptavidin-HRP Conjugate, the ratio of HRP and Streptavidin covalent cross-linking is greater than 3, which is more convenient and more sensitive than Streptavidin and Biotin-HRP conjugate for detection.Streptavidin with lower non-specific binding than avidin is used to make the detection result lower background and more sensitive.This kit does not provide reagents related to biotin probe labeling. For the preparation of biotin-labeled DNA probes or EMSA probes, the biotin 3' end DNA labeling kit or EMSA produced by aladdin can be used accordingly. Probe Biotin Labeling Kit .This kit is sufficient for at least 10 10×10cm membranes with biotin-labeled EMSA probes, a total of 1000cm2.

Precautions：

Positively charged nylon membranes and reagents required for gel electrophoresis are required but not supplied in this kit. Positively charged nylon membrane can be ordered from . The Blocking Buffer or the Wash Buffer can be ordered separately from when more buffer is needed.ECL Moon reagent A and reagent B are hazardous. Please take effective measures to avoid direct contact or inhalation.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instructions for Use：

1. In the case of using Biotin-labeled probes, this kit can be used to nucleic acid detection after completion of Southern/Northern hybridization or after transfer and cross-linking of RPA or EMSA. The amount of reagents used in the following procedure is for a 10x10cm membrane. For different size of membranes, the amount of each reagent should be adjusted proportionally.2. Dissolve the Blocking Buffer and the Wash Buffer in a water bath at 37-50℃.Note: The Blocking Buffer and the Wash Buffer must be completely dissolved and mixed well prior to use. ,These two reagents can be used at temperatures between 20-50℃.3. Add 15ml of Blocking Buffer into a suitable container, transfer the cross-linked nylon membrane into the buffer, and then incubate for 15 minutes with slow shaking on a shaker.4. Add 7.5µl of Streptavidin-HRP Conjugate into 15ml of Blocking Buffer, and mix well.5. Transfer the membrane from the Blocking Buffer into the Blocking Buffer containing Streptavidin-HRP Conjugate prepared in the previous step. Incubate for 15 minutes with slow shaking on a shaker.6. Prepare 125ml of wash buffer by mixing 25ml of Wash Buffer (5X) with 100ml of ddH2O or Milli-Q pure water.7. Transfer the membrane into 15-20ml of wash buffer and rinse for 1 minute.8. Remove the wash buffer, add 15-20ml of new wash buffer, and wash for 5 minutes with agitation on a shaker.9. Repeat step 8 three times (four washes in total). Wash for 5 minutes each time.10. Transfer the membrane to another container with 20-25ml of Equilibration Buffer, and incubate for 5 minutes with slow shaking on a shaker.11. Prepare the ECL Moon working solution by mixing 5ml of ECL Moon reagent A and 5ml of ECL Moon reagent B. Note 1: The ECL Moon working solution must be prepared freshly and used immediately. Note 2: From this step onwards, the operation and precautions are the same as those of the fluorescence assay during Western blot.12. Take the membrane and remove excess liquid with absorbent paper. Immediately place the membrane with sample side up in a clean container or on a plastic wrap.13. Carefully add the ECL Moon working solution prepared in step 11 on the surface of the membrane and make sure that the membrane is completely covered with the working solution. Leave at room temperature for 2-3 minutes.14. Take the membrane and remove excess liquid with absorbent paper. Place the membrane between two pieces of clear plastic wrap, and put it in a film cassette with the sample side facing up.15. In a dark room, place a X-ray film on top of the membrane, and expose for 1-5 minutes. The exposure time can be adjusted based on the exposure result. The film can be exposed directly for 30 seconds, 1, 3, 5 minutes or more, and then developed together to obtain an ideal result.FAQ:1. The background is high.It may be due to precipitation or turbidity in the Blocking Buffer or Wash Buffer. Please make sure the Blocking Buffer and the Wash Buffer should be completely dissolved before use.2. A speckled background appears.It may be caused by precipitation in the Streptavidin-HRP Conjugate due to long-term storage. Centrifuge the solution at 12000-14000×g for 1min prior to use. Air bubbles between membrane and gel during transfer can also cause a speckled background. So please make sure that there are no air bubbles between the gel, membrane and filter paper for transfer.3. No band or weak signal.a. The labeling efficiency is too low. The solution is to examine and improve the labeling efficiency.b. The amount of probe or sample is insufficient. The solution is to increase the amount of probe or sample.c. The nucleic acid to be detected is degraded. The solution is to prepare a new sample without nucleic acid degradation.d. The membrane transfer is not efficient. The solution is to check the method and procedure for membrane transfer. It is better to load a pre-stained standard as a control for membrane transfer.e. An inappropriate membrane is selected. Although nitrocellulose membrane can also be used in many cases, the positively charged nylon membrane is preferred.f. The membrane dries out during the detection. The membrane should be covered completely by liquid and remain wet throughout the detection process.g. There is no cross-linking or the cross-linking effect is not good. The solution is to carry out appropriate cross-linking and check the cross-linking effect.h. The Wash Buffer (5X) is used directly without dilution. Please dilute the Washing Buffer (5X) to 1X before use.i. The exposure time of X-ray film is insufficient. The solution is to appropriately extend the exposure time.