DNA Pull Down Kit(Animal),Absin_specification/price/image

Product Detail
Company Profile

Product Specification

Usage

A, Total protein extraction

1. Cell samples

(1) washing: precooling 1 ml of PBS washing sample (about 2 x 107 cells) 2 times, the last blot PBS as far as possible;

(2) pyrolysis: according to the amount of cells to join 980 ul Lysis buffer, 10 ul Protease inhibitors (100 x) fully, the ice cracking 30 min, once every 5 min vortex, 10 s/time;

(3) Using ultrasonic cell breaker for 5min, power 20%, ultrasonic 3s, intermittent 3s, ice bath ultrasound;

(4) the centrifugal: 4 ° C, 12000 RPM, 10 min, collecting supernatant.

2, tissue samples

(1) grinding: take fresh organizations or low temperature (about 0.3 g), put in precooling of mortar, ground into a powder in liquid nitrogen, put to a new EP in the tube;

(2) Lysis: Take 980 ul Lysis buffer, 10 ul Protease inhibitor (100 x) blending as cracking fluid, absorb 800 ul cracking liquid to join the mortar, continue to grind on the ice samples into 5-10 min to the exquisite homogenate, transferred to the new EP in the tube, To add the remaining 190 ul mortar cracking fluid to collect the remaining samples, also transferred to the EP tube;

(3) is equipped with sample homogenate of EP tube fully on the ice cracking 30 min, every 5 min vortex, 10 s/time;

(4) using ultrasonic cell disruptor ultrasonic 8 to 10 min, power 20%, ultrasonic 3 s, intermittent 3 s, ice bath ultrasonic;

(5) the centrifugal: 4 ℃, 12000 RPM, 10 min, collecting supernatant, clear up adding Lysis buffer added volume to 1 ml, blending.

Note: The whole process of protein extraction is operated on ice to reduce protein degradation caused by high temperature. It is better not to have bubbles during the ultrasound process to reduce protein degradation. After cracking of total protein in - 20 ℃.

B. Preparation and washing of magnetic beads

(1) the Nucleic Acid - Compatible Streptavidin Magnetic Beads from 4 ° C, freezer upside down blending Magnetic bead storage solution for many times, respectively take 30 ul to 2 1.5 mL EP in the tube, as the control group and experimental group, The beads were placed on a magnetic rack for 1min to separate the beads, and the supernatant was discarded.

(2) in the control group and experimental group with 500 ul Nucleic dilution buffer, heavy suspension magnetic beads, placed in a magnetic rack 1 min, abandon supernatant, repeat the step 3 times.

C, Magnetic beads bound to DNA

(1) the test tube to join 300 pmol biotin labeled DNA probe, to take care of without biotin labeled DNA plus amount or not, with Nucleic dilution buffer added volume to 500 ul, quiet room temperature 2 h incubation on mixing apparatus;

(2) to take care of and the experimental pipe from the mute mixing apparatus, placed in a magnetic rack let stand for 1 min, abandon the supernatant;

(3) 500uL Nucleic dilution buffer was added to the control and experimental tubes, the magnetic beads were resuspended, placed on the magnetic frame for 1min, and the supernatant was discarded. This step was repeated for 3 times.

D. Dna-bead binding protein

Tube (1) to take care of and experiment with 450 ul, extraction of Protein with Protein dilution buffer added to 1 ml volume, mute mixing apparatus at 4 ° C overnight incubation (about 16 h), the reserved 100 ul cracking liquid as Input group;

(2) The control tube and experimental tube were removed from the silent mixer, placed on the magnetic frame for 1min, and the supernatant was discarded;

(3) with 1 ml Protein dilution buffer, heavy suspension magnetic beads, placed in a magnetic rack let stand for 1 min, abandon supernatant, repeat the step 5 times.

E. Elution of the complex

Tube (1) to take care of and experiment in 100 ul Elution buffer, blending a boiling water bath after 8 to 10 min, placed in a magnetic rack let stand for 2 min, take on to new EP in the tube, to pull down the product, marked as the control group and experimental group, 2 added 20 ul to each tube 6 x Loading buffer, boiling water bath 8-10 min;

(2) the reserved Input 100 ul cracking liquid, joined 20 ul 6 x Loading buffer, boiling water bath 8-10 min;

(3) the control group, experimental group and the Input - 20 ° C saved for later use. Subsequently, silver staining, mass spectrometry or WB detection were performed.

Synonym

DNA pull down kit

Description

I. Experimental principle

DNA pull down technology is a powerful tool for studying DNA-protein interaction in vitro. In this technique, specific DNA probes are designed for the target region and labeled with desulfuryl biotin. After labeling, the labeled probes can bind to the streptavidin conjugated to the magnetic beads, and then incubated with the total protein extract. The non-specific binding proteins were removed by washing, and then the target DNA probe-protein complexes were obtained by elution. Finally, the protein types were identified by Western Blot or mass spectrometry (MS). The schematic diagram is as follows:

2, experiment flow chart

Composition

Name	Size（6T）	Storage
Lysis buffer	9mL	4°C
Protease inhibitor(100×)	35uL	-20°C
Nucleic dilution buffer	30mL	4°C
Protein dilution buffer	45mL	4°C
Nucleic-Acid Compatible Streptavidin Magnetic Beads	200uL	4°C
Elution buffer	800uL	Avoid light at 4°C

General Notes

Applicable to cells, animal tissues

Storage Temp.

Store at -20 & 4 ° C (see component storage information for details); Valid for 1 year

AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.