Pulldown: DNA/RNA

  
        

【Pulldown Kits and Price】

Type Catalog# Pack Price$ Description
DNA-Prot.
Pulldwon
see table 2 10
20
421
571
Including DNA binding protein Pulldown reagent, purified protein gel electrophoresis reagent, gel dyeing reagent and detailed operating instructions (shipped with reagent).
RNA-Prot.
Pulldwon
see table 2 10
20
507
650
Including RNA binding protein Pulldown reagent, purified protein gel electrophoresis reagent, gel dyeing reagent and detailed operating instructions (shipped with reagent)
Note: (1) order must be added after the catalog number. (2) Viagene also offers DNA-protein with RNA-protein pulldown service (view).

DNA_pulldown

【Principle】

The DNA-protein Pulldown / Pull-down kit is used to purify DNA-binding proteins from tissue and cell samples. The priciple is as follows:

Streptavidin beads can bind to biotin-labeled DNA / protein complexes. Separating the conjugate from the supernatant by centrifugation or by centrifugation, discarding the supernatant and washing the beads, and then denaturing the protein with the SDS sample solution to separate the beads from the magnetic beads. The proteins in the separation solution were separated by SDS polyacrylamide gel electrophoresis (PAGE) or two-dimensional electrophoresis, and SDS PAGE was stained with high sensitivity Coomassie brilliant blue (CBB). The desired protein bands were then excised from the stained gel for mass spectrometry to confirm the protein type and type.

The main steps are shown in Figure 1 and Figure 2 on the right.

【Features】

1. Combination of specific specificity, non-specific binding less.
2. SDS PAGE staining was clear and sharp.
3. High staining sensitivity, can be purified a small amount of DNA binding protein (Figure 3).
4. Pulldown protein is suitable for mass spectrometry.

[Kit]

1. Chain avidin beads.
2. Biotin-labeled DNA probe.
3. DNA-protein or RNA-protein binding reaction.
4. DNA or RNA affinity purification lotion.

5. SDS sample buffer

pulldown_SDSgel

6. SDS PAGE Staining solution

7. SDS PAGE Destaning solution

8. User' manual

【Other equipment and materials needed】

参考产品使用说明书

【Applications】

1. Identification of unknown DNA / RNA binding proteins; in some cases, it may not be known which proteins are bound to DNA / RNA and what proteins are. Use of this kit combined with mass spectrometry to identify unknown binding proteins without the use of purified proteins or antibodies.

2. Identification of DNA-binding protein components; many transcription factors are multimers of multiple proteins. For example, NFkB has P65, P52, P50, C-rel And Rel-B 5 subunits. Activation of NFkB is the same or different subunit between the dimers, identified NFkB subunits commonly used Supershift EMSA, the need for the use of antibodies for a variety of NFkB subunits, expensive. Use the kit to replace NFkB subunit with Supershift EMSA identifies the subunits of the DNA-protein complex without the use of any antibodies.

3. Identification of the interaction of DNA-binding proteins with other proteins; Some transcription factors combined with DNA also require some involvement with DNA-binding proteins. The use of this product can simultaneously DNA binding protein and non-DNA binding protein identification.

【Notes】

1. This reagent is able to interface seamlessly with Viagene's EMSA kit. However, if you use Thermo, Signosis or Phenon Day EMSA reagents, may not achieve the desired results or lead to complete failure. The reason is because different manufacturers use the composition of the reaction solution is different from the different components of the reaction solution on the same transcription factor determination will appear different results (see Figure 4).

2. Before using this product, it is recommended to carry out routine EMSA determination. The EMSA assay can provide the following information;

1)There is no DNA and protein binding in the EMSA assay. Significant EMSA binding bands indicate good identification results.

2)The strength of the EMSA binding band suggests the reaction volume of Pulldown. The binding volume is small and the volume of the sample nucleoprotein is small.

3)There is no nonspecific band or high background in the EMSA assay. If the nonspecific band is clear, the negative control of the same cell line needs to be used to determine the nonspecific bandage after purification.

3. Please follow the operating instructions in strict accordance with the operation manual. If you encounter problems, use biotin to mark the quality control probe with the nucleoprotein sample for Pulldown Experiment. The DNA binding protein determined by the biotin-labeled control probe is present in almost all nuclei. The supernatant of Pulldown should be seen as a protein band in the gelatin stained with Coomassie Brilliant Blue. Negative results suggest 1) the nuclear protein in the sample degrades the nucleoprotein, 2) the total amount of nucleoprotein used for pulldown is not enough, 3) the nucleoprotein concentration in the sample is too thin, and 4) the nucleoprotein is lost during the extraction process, suggesting that the nucleoprotein sample needs to be reconstituted The EMSA of the binding protein was identified.

PAGE_Stain_compare  pulldown_bands

 Pic. 3, 20ul, 10ul, 5ul, 2.5 ul, 1.25ul cell lysate PAGE glue. After electrophoresis, the gel plates were cut into two pieces, stained with Thermo's Coomassie Brilliant Blue (CBB), respectively.
Pic. 4, With streptavidin beads DNA-binding protein: 1, from the EMSA determination of the positive sample pulldown unknown binding protein, 2, EMSA determination of negative samples of the pulldown results. Lane 2 uses the same cell line as lane 1, but the cells are not activated. 3 and 4 for NFkB EMSA Positive and negative samples of pulldown results. Combined with mass spectrometry, subunits of NFkB can be identified. 5, protein molecules Marker. 6 with 7, sample with pulldown operation same as lanes 3 and 4. The Pulldown protein was analyzed by SDS-PAGE and stained with Bi * CBB.

  

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