DYKDDDDK-Nanoab-Magnetic beads

La description

DYKDDDDK-Nanoab-Magnetic beads CAT：FNM*******0 Price: $**3 for *0T(negotiable) product ID: FNM
Storage conditions:Stored at **0℃ for one year(to ensure full sealing), avoid centrifugation, drying and freeze-thaw.
 
Product description
Magnetic nanospheres of coupling anti-DYKDDDDK-tag nanoantibodies are used for immunoprecipitation of DYKDDDDK-tag fusion protein
Product advantages
No heavy chain and light chain, clean background;
Ready-to use type, saving time;
High affinity and high binding ability.
Scope of application
It can be used for immunoprecipipation(IP)/immunocoprecipipation(CoIP)、chromatin immunoprecipitation(ChIP)/RNA-binding protein immunoprecipipation(RIP), enzyme activity determination, mass spectrometry analysis, etc.
Specificity
The specific binding is located at the N-terminal, C-terminal and internal DYKDDDDK-tag of the fusion protein.
Product features
Storage buffer: PBS(containing *0%  ethanol);
Storage conditions: Stored at 4℃ for one year(to ensure full sealing), avoid centrifugation, drying and freeze-thaw.
 
Experimental steps:
Plant tissue cracking treatment:
Take an appropriate amount of plant tissue samples (leaves, etc.) and place them in frozen liquid nitrogen, and then put the frozen plant tissue samples in a mortar for grinding, so as to fully grind and destroy their cell walls. Add *******0ul RIPA lysate (added with protease inhibitor PMSF, etc.) for cracking. In order to improve the cracking efficiency, add **0ul glass powder and fully shake for *0min. After cracking, ****0 rpm, centrifuge for *0min, suck the supernatant and place it in a new centrifuge tube, and discard the sediment.
Collect cells
Approximately ******7 cells expressing DYKDDDDK-tag fusion protein were used for each immunoprecipitation reaction, and the number of cells could be appropriately adjusted according to the expression of DYKDDDDK-tag fusion protein. Suck out the medium and add precooled 1 × PBS, rinse twice, collect adherent cells by cell scraping or trypsin digestion, transfer the cells to a centrifuge tube, centrifuge **0 g for 3 minutes and discard the supernatant.
 
Cell lysis
1. Protease inhibitors are added to the lysis buffer, and the cells are suspended with a pre-cooled lysis buffer of **0μl.
2. Put it on the ice for *0 minutes and blow it fully every *0 minutes.
3.Centrifuge ****0 g at 4 ℃ for *5 minutes, transfer the cracking product (supernatant) to a new precooling tube, and discard the precipitation.
Note: At this time, the cell lysis product can be preserved at **0°C for a long time.
Balanced beads
4. Fully mix DYKDDDDK-Nanoab-Magnetic Beads, absorb *0μl of the product into the **0μl precooled cracking buffer, separate the magnetic beads on the magnetic frame until the top becomes clear, and discard the liquid.( This step is optional)
Binding protein
5. The balanced DYKDDDDK-Nanoab-Magnetic Beads are added to the cell lysis product (*0μl can be added directly to the cell lysis product if step 4 is not done) and rotate and combine at 4°C for 1 hour. The combination time can be adjusted according to the needs of the experiment. If necessary, the *0μl cracking product is retained for immunoprinting analysis.
6. Separate the magnetic beads on the magnetic shelf until the upper clear becomes clear and discard the upper liquid. If necessary, *0μl supernatant is retained for immunoprinting analysis.
Wash the beads
7. Use **0 μL precooled pyro lysis buffer resuspended DYKDDDDK-Nanoab-Magnetic Beads, separated the magnetic beads on the magnetic shelf until the supernatant became clear, discarded the supernatant and washed again for 3 times. Minimize cleaning time.
Elution protein
Method 1:
8. Adding *0μl 2×SDS-sample buffer suspends DYKDDDDK-Nanoab-Magnetic Beads.*5 ℃, heated for *0 min, fully denatured, separated on the magnetic shelf and the collected products can be analyzed by SDS-PAGE and immunoblotting
Method Two:
9. Add *0μl 0.2M pH2.5 glycine to elute the bound protein for a *0 second incubation period, during which it is continuously mixed, isolated and collected on the magnetic shelf, and immediately add 5μl 1.0 M Tris (pH*0.4) to neutralize the acidic glycine.
Note: This step can be repeated to improve the efficiency of elution.
Method Three:
*0. Add *0μM DYKDDDDK-peptide for elution.
 
Optional experimental scheme
Option 1:
If the activity of DYKDDDDK tag fusion enzyme needs to be tested, it can be detected directly without elution.
Option 2:
If chromatin immunoprecipitation (ChIP) experiments are needed, they are mainly used for protein/DNA interactions containing DYKDDDDK fusion proteins. Chromatin immune precipitation generally includes cell fixation, chromatin fracture, chromatin immune precipitation, reversal of  linkage reactions, DNA purification and DNA identification. Among them, the early cell is fixed, and the chromatin fracture remains unchanged; then go directly to step 5 of the instruction manual. After adding the cell lysis product, the DNA-protein complex binds to DYKDDDDK-Nanoab-Magnetic Beads;
Enter steps 6 and 7, isolate the complex; enter step 9, get the eluted complex; reverse the later crosslinking reaction, DNA purification and DNA identification are equivalent to ordinary ChIP experiments. The experimental steps of RNA-binding protein immunoprecipitation (RIP) are the same as above.
Option 3:
DYKDDDDK-Nanoab-Magnetic Beads can not only detect and verify the interaction between proteins in vivo and outside, but also combine mass spectrometry to screen unknown proteins that interact with known proteins. The operation step is the same as the immune precipitation method. After obtaining the eluted complex, then SDS-PAGE analysis is carried out. After staining by Coomas bright blue or silver staining, the strips of unknown proteins are cut off, and unknown proteins are identified by mass spectrometry.