Application
Immunoprecipitation
Immuno-precipitation step
Protein G
Sample
Human Cell lysate - whole cell (Human Embryonic Kidney 293 cells)
Specification
Human Embryonic Kidney 293 cells
Total protein in input
1000 µg
Other product details
Incubation time
12 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: Non-denaturing lysis buffer
Western Blot antibody
Name
Non-Abcam antibody was used: Histone H3
Host species: Human
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Human
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Additional data
Additional Notes
methylated Lysine was immunoprecipitated using 1mg Human Embryonic Kidney 293 cells whole cell extract, 1´g of it polyclonal to methylated Lysine and 30´l of protein G magnetic beads (+). Normal rabbit IgG antibody was added as control. Cells were lysed by brief sonication in co-immunoprecipitation buffer (Non-denaturing lysis buffer) supplemented with protease inhibitors cocktails (Roche). Lysates were centrifuged for 30 min at 14,000 ¿ g and the resulting supernatant was precleared by incubation with Protein G. The precleared supernatant was subjected to immunoprecipitation using methylated Lysine or control IgG antibodies at 4¯C for 3 h, protein complexes were collected by incubation with Protein G for 12 h at 4¯C. Proteins were eluted by addition of 10´l SDS loading buffer and incubated for 10min at 95¯C sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with anti-Histone H3 (purchased from GENETEX:GTX122148). Secondary: Clean blot (HRP conjugate) at 1/5000 dilution. Band: 17kDa: methylated-Lysine histone H3.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Dr. Yi-Wen Chang
Verified customer
提交于 Jan 26 2015