Immunoblot analysis was performed using standard procedures, with a 1:5,000 −1:6,666 dilution of α-FzlA primary antibody [21 (link)], a 1:50,000 dilution of α-HU primary antibody [60 (link)], and/or 1:10,000 of HRP-labeled α-rabbit secondary antibody (PerkinElmer) on nitrocellulose membranes. Chemiluminescent substrate (PerkinElmer) was added to facilitate protein visualization via an Amersham Imager 600 RGB gel and membrane imager (GE).
Amersham imager 600 rgb gel and membrane imager
Manufactured by GE Healthcare
The Amersham Imager 600 RGB is a gel and membrane imager designed for imaging proteins, nucleic acids, and other biomolecules. It uses a high-resolution color CCD camera and RGB LED illumination to capture images of stained gels and blots. The imager provides quantitative analysis capabilities and supports a range of sample types and imaging techniques.
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5 protocols using amersham imager 600 rgb gel and membrane imager
1
Immunoblot Analysis of FzlA and HU
2
Western Blotting of Protein Expression
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Western blots were performed using standard lab procedures. Log phase cultures were lysed in SDS-PAGE loading buffer and boiled for 10 minutes. Equivalent OD units of cell lysate were loaded. Standard procedures were followed for SDS-PAGE and protein transfer to nitrocellulose membrane. Antibodies were used at the following concentrations: Primary antibodies used were Flag-M2 – 1:1,000 (Sigma, St. Louis, Missouri) and CdnL −1:2,500 (33 (link)). Secondary antibodies used were 1:10,000 of HRP-labeled α-mouse (for Flag) (PerkinElmer) or α-rabbit (PerkinElmer) (for CdnL). Chemiluminescent substrate (PerkinElmer) was added to visualize proteins via an Amersham Imager 600 RGB gel and membrane imager (GE).
3
Immunoblotting and co-immunoprecipitation protocol
4
Immunoblotting of Whole Cell Lysates
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For immunoblotting of whole cell lysates, log phase cells were pelleted, then resuspended and boiled in SDS‐PAGE loading buffer. For co‐immunoprecipitation, eluted samples were boiled in SDS‐PAGE loading buffer. Samples were resolved by SDS‐PAGE, and protein was subsequently transferred to nitrocellulose membranes. Membranes were first probed with α‐FzlA (Goley et al., 2010b ) (1:8000), α‐HU (Bowman et al., 2010 ) (1:50 000; loading control), α‐FtsZ (1:20 000) or α‐RFP (1:2000) primary antibodies, then incubated with HRP‐conjugated α‐rabbit (1:20 000) secondary antibody, or probed with α‐FLAG (Sigma) (1:1000) primary antibody, then incubated with HRP‐conjugated α‐mouse (1:10 000) secondary antibody. Membranes were subsequently imaged with an Amersham Imager 600 RGB gel and membrane imager (GE).
5
Immunoblotting Techniques for Bacterial Proteins
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Equivalent OD units of cell lysate were loaded on an SDS-PAGE gel following cell harvest by centrifugation, lysing 1X SDS loading dye, and boiling for 5 – 10 minutes. SDS-PAGE and protein transfer to nitrocellulose membranes were followed using standard procedures. Antibodies were used at the following dilutions: CdnL 1:10,000 (14 (link)); MreB 1:10,000 (Regis Hallez, University of Namur); SpmX 1:20,000 (Patrick Viollier, University of Geneva); GFP 1:2,000 (Clonetech Labs Catalog #NC9777966); HRP-labeled α-rabbit secondary 1:10,000 (BioRAD Catalog #170-6515); and/or HRP-labeled α-mouse secondary (Cell Signaling Technology Catalog #7076S). Clarity Western Electrochemiluminescent substrate (BioRAD Catalog #170-5060) was used to visualize proteins on an Amersham Imager 600 RGB gel and membrane imager (GE).
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