HeLa nuclear extract was prepared as previously described [13 (link)]. Extracts were incubated with Protein A-Dynal beads coupled with polyclonal antibody against human RECQ1 (A300-450A, Bethyl Lab), FEN-1 (A300-255A, Bethyl Lab) or normal rabbit IgG (Vector Labs) at 4°C for 90 min, and the immunecomplexes were eluted with 2x SDS-sample buffer following three washes with lysis buffer. Where indicated, nuclear extract was pre-incubated with benzonase (Sigma, 50 U/ml, 2 h at 4°C), a general endonuclease for DNA and RNA. Proteins were resolved by 8-16% SDS-PAGE, transferred onto PVDF membrane and subjected to Western detection of RECQ1 (1:750, sc-25547, Santa Cruz Biotech), FEN-1 (1:1000, Bethyl Lab).
Protocol detail
Immunoprecipitation of RECQ1 and FEN-1
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Corresponding Organization :
Other organizations : Howard University, Georgetown University Medical Center, Georgetown University, University of Nevada, Las Vegas
Top 5 similar protocols
Variable analysis
independent variables
- Incubation of HeLa nuclear extract with Protein A-Dynal beads coupled with polyclonal antibody against human RECQ1 (A300-450A, Bethyl Lab) or FEN-1 (A300-255A, Bethyl Lab)
- Pre-incubation of nuclear extract with benzonase (Sigma, 50 U/ml, 2 h at 4°C), a general endonuclease for DNA and RNA
- Presence and levels of RECQ1 and FEN-1 proteins in the immunecomplexes, as detected by Western blotting
- Incubation of HeLa nuclear extract with Protein A-Dynal beads coupled with normal rabbit IgG (Vector Labs)
- Positive control: Incubation of HeLa nuclear extract with Protein A-Dynal beads coupled with polyclonal antibody against human RECQ1 or FEN-1
- Negative control: Incubation of HeLa nuclear extract with Protein A-Dynal beads coupled with normal rabbit IgG
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!