magnetm halotag® magnetic affinity beads  (Promega)

Journal: PLoS ONE

Article Title: WDR76 Co-Localizes with Heterochromatin Related Proteins and Rapidly Responds to DNA Damage

doi: 10.1371/journal.pone.0155492

Figure Lengend Snippet: (A) Heat map of relative abundance of 40 proteins shared between WDR76 and histones. The color intensity represents protein abundance with bright yellow displaying highest abundance (average distributed spectral counts) and black indicates that the protein was not detected in a particular purification. (B) Western blots of inputs and purifications from Halo-WDR76 stable cell line or HEK293FRT parental cell line shows that PARP1 and XRCC5 can be co-purified with WDR76, which is consistent with the proteomics analysis. Lane 1 and 2 are whole cell lysates from Halo-WDR76 stable cell line or HEK293FRT cells. Lane 3 and 4 are the protein purified with HaloTag™ magnetic beads from Halo-WDR76 stable cell line or HEK293FRT cells. The top panel was blotted with anti-PARP1 and anti-Actin antibodies; the middle panel was blotted with anti-XRCC5 and anti-Actin antibodies; the last panel was blotted with anti-WDR76 and anti- HaloTag™, which was not detected because of the cleavage during elution. (C) Topological data analysis was performed on 164 proteins with a Z-score greater than 2 in WDR76 and at least one histone purification compared to controls. All the nodes in the network are colored based on metric PCA coordinate 2. Filters with variance normalized Euclidean metric were used (resolution 30, gain 3.0x).

Article Snippet: MagneTM HaloTag ® magnetic affinity beads and HaloTagTMTMRDirect ligand were purchased from Promega (Madison, WI).

Techniques: Quantitative Proteomics, Purification, Western Blot, Stable Transfection, Magnetic Beads

Journal: PLoS ONE

Article Title: WDR76 Co-Localizes with Heterochromatin Related Proteins and Rapidly Responds to DNA Damage

doi: 10.1371/journal.pone.0155492

Figure Lengend Snippet: Cells stably expressing Halo-WDR76 were separately transiently transfected with CLIPtag™-FLAG proteins and HaloTag™ protein purifications conducted. Western blotting for tubulin was used as a loading control and western blotting with an anti-FLAG antibody was used to detect CBX1 (A), CBX3 (B), and CBX5 (C). Both the inputs and the results of the pulldown is shown. (D) Cells stably expressing Halo-WDR76 were transiently transfected with CLIPg™-FLAG construct to demonstrate that this construct was not responsible for the co-purifications of CBX proteins with WDR76.

Article Snippet: MagneTM HaloTag ® magnetic affinity beads and HaloTagTMTMRDirect ligand were purchased from Promega (Madison, WI).

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Control, Construct