FACS analysis of mesenteric lymph node cells: CD4-APC (eBioscience, clone GK1.5), TCRb-PE (eBioscience, clone H57-597), CD69-biotin (eBioscience, clone H1.2F3), Streptavidin-FITC, (eBioscience). Immunohystochemistry: F4/80 (AbD Serotec; MCA497). Western blot: SIRT2 (H-95, SantaCruz sc-20966), Acetyl-NF-κB (Acetyl-K310, Abcam, ab19870), phospho-IKbα (ser32/36, Cell Signaling 9246), IKbα (L35A5, Cell Signaling 4814), Hsp90 (BD Transduction Laboratories, 610418).
Sirt2 h 95
Manufactured by Santa Cruz Biotechnology
SIRT2 (H-95) is a recombinant protein that corresponds to amino acids 1-95 of human SIRT2. SIRT2 is a member of the sirtuin family of NAD+-dependent protein deacetylases.
Automatically generated - may contain errors
2 protocols using sirt2 h 95
1
FACS, IHC, and Western Blot Analysis of Mesenteric Lymph Node Cells
2
Immunofluorescence Assay for SIRT1 and SIRT2
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells were seeded in eight-well chamber slides and treated for 72 h. Media was
removed and cells were rinsed with PBS. Cells were fixed with 4%
paraformaldehyde for 15 min at room temperature followed by rinsing with PBS
(three times, 5 min each). The cells were then permeabilized using ice-cold
methanol and rinsed again with PBS as previously described. The cells were
blocked with 5% BSA solution for 1 h, followed by rinsing with PBS again as
previously described. Subsequently, 200 µl of primary antibodies: SIRT1 (B-10)
(Santa Cruz, Cat#sc-74504, mouse monoclonal) and SIRT2 (H-95) (Santa Cruz,
Cat#sc-20966, rabbit polyclonal) were added to the cells and incubated at 4°C
overnight. Next, the cells were rinsed with PBS followed by incubation with
secondary antibodies (anti-mouse or anti-rabbit IgG, highly cross-adsorbed
secondary antibody, Alexa Fluor, Thermo Fisher, USA) for 1 h at room
temperature. Cells were washed in PBS three more times (5 min each). The slides
were removed from the chamber casket and mounted using Fluoroshield with DAPI
mounting media (Sigma). Cells were viewed and imaged using an inverted
fluorescent microscope (BX41, Olympus).
removed and cells were rinsed with PBS. Cells were fixed with 4%
paraformaldehyde for 15 min at room temperature followed by rinsing with PBS
(three times, 5 min each). The cells were then permeabilized using ice-cold
methanol and rinsed again with PBS as previously described. The cells were
blocked with 5% BSA solution for 1 h, followed by rinsing with PBS again as
previously described. Subsequently, 200 µl of primary antibodies: SIRT1 (B-10)
(Santa Cruz, Cat#sc-74504, mouse monoclonal) and SIRT2 (H-95) (Santa Cruz,
Cat#sc-20966, rabbit polyclonal) were added to the cells and incubated at 4°C
overnight. Next, the cells were rinsed with PBS followed by incubation with
secondary antibodies (anti-mouse or anti-rabbit IgG, highly cross-adsorbed
secondary antibody, Alexa Fluor, Thermo Fisher, USA) for 1 h at room
temperature. Cells were washed in PBS three more times (5 min each). The slides
were removed from the chamber casket and mounted using Fluoroshield with DAPI
mounting media (Sigma). Cells were viewed and imaged using an inverted
fluorescent microscope (BX41, Olympus).
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