Fixation was carried out with ice-cold 4% paraformaldehyde (30 min), followed by permeabilization when necessary with 0.1% Triton X-100 on ice (10 min) and blocking in 3% bovine serum albumin (BSA;30 min). Incubation with primary antibodies was carried out in a 3% BSA solution (overnight) at 4 °C. Primary antibodies used were: rabbit anti-SIRT1(H300) (1:400; Santa Cruz, Cat. No. sc-15404), rabbit anti-NF-kB p65 (1:400; Thermofisher, Cat. No. PA1-186), mouse anti-HIF-1α(H1alpha-67) (1:80; Santa Cruz, Cat. No. sc-53546). After washing, cells were incubated with secondary antibodies for 45 min RT, washed, and mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution (both from Sigma). Secondary antibodies used were: Alexa-Fluor 546-anti-mouse (1:300; Invitrogen), Alexa-Fluor 488-anti-rabbit (1:300; Invitrogen). Digital images were captured with a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss, Oberkochen, Germany).
Rabbit anti sirt1 h300
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Rabbit anti-SIRT1(H300) is a primary antibody that specifically detects SIRT1 (Sirtuin 1) protein in various sample types. It is produced in rabbits and recognizes an epitope located within the first 300 amino acids of the human SIRT1 protein sequence.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole dapi containing mounting solution, by Merck Group (2 mentions)
Apotome 2 acquisition system, by Zeiss (2 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Observer z1 microscope, by Zeiss (1 mentions)
Anti mouse alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Mycophenolic acid mpa, by Merck Group (1 mentions)
Mouse monoclonal anti his, by Merck Group (1 mentions)
Inside fix solution, by Miltenyi Biotec (1 mentions)
Inside perm solution, by Miltenyi Biotec (1 mentions)
Anti rabbit af488, by Thermo Fisher Scientific (1 mentions)
Anti mouse af488, by Thermo Fisher Scientific (1 mentions)
5 protocols using rabbit anti sirt1 h300
1
Immunofluorescence Staining of SIRT1, NF-kB, and HIF-1α
2
Characterizing Inauhzin Compounds in Cancer Cells
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Human lung carcinoma H460 and human colon cancer HCT116 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (PBS), penicillin, and streptomycin. Inauhzin (INZ), Inauhzin inactive analogue INZ (O) (INZ9 in [Zhang et al., 2012a (link)]) and Biotinylated INZs were synthesized and characterized by NMR and LC-MS as described (Zhang et al., 2012b (link)). The purity of the compounds is higher than 90%. Mycophenolic acid (MPA) was purchased from Sigma-Aldrich (St.Louis, Missouri). Mouse monoclonal anti-p53 (DO-1), rabbit anti-p21 (M19), mouse anti-p21 (F5), rabbit anti-SIRT1 (H300) and goat anti-RPL11 (N17) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas). for immunoblotting. Cleaved PARP, PARP (9542), Puma were from Cell Signaling Technologies. Mouse anti-MDM2 (2A10), rabbit anti-RPL11 and anti-RPL5 antibodies were described previously (Zeng et al., 1999 (link); Sun et al., 2008 (link)). Antibodies for immunostaining were rabbit polyclonal anti-p53 (FL-393; Santa Cruz) and monoclonal nucleostemin antibodies (Chemicon, Billerica, Massachusetts).
3
Immunoblot Analysis of NQO1 and SIRT1
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Cell extracts and immunoblot analysis were carried out as previously described (Tsvetkov et al., 2011 (link)). The antibodies used were: goat anti NQO1 C19 and R20 (Santa Cruz), rabbit anti SIRT1 H300 (Santa Cruz) and mouse monoclonal anti His (Sigma).
4
Immunofluorescent Analysis of SIRT1 and NF-κB
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Cells were fixed using InsideFix Solution (Miltenyi Biotec, Bologna, Italy) and incubated overnight at 4 °C with primary antibodies diluted in InsidePerm solution (Miltenyi, Bologna, Italy). The antibodies used were rabbit anti-SIRT1(H300) (1:400; Santa Cruz Biotechnologies, Santa Cruz, CA, USA, Cat. No. sc-15404) and rabbit anti-acetyl-NF-kB p65 (Lys310) (1:30; Cell Signaling, Danvers, MA, USA, Cat. No. 3045). After washing, cells were incubated with secondary antibodies, diluted in InsidePerm solution, for 45 min RT. The secondary antibodies used were AF488-anti-mouse (1:300; Thermofisher Scientific, Waltham, MA, USA) and AF488-anti-rabbit (1:300; Thermofisher Scientific, Waltham, MA, USA). After washing, slides were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution (Sigma-Merck, Darmstadt, Germany). Digital images were captured with a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss, Oberkochen, Germany). The number of immunopositive cells with nuclear SIRT1 was determined by cell counting in at least five randomly selected fields/well.
5
Purification and Co-IP of NQO1 and SIRT1
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pET28-His-TEV-NQO1 and pET28-His-SIRT1 were expressed in bacteria and cells were lyzed by sonication in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, Lysozyme and 1 mM PMSF. Soluble His-TEV-NQO1 and His-SIRRT1 were purified using Ni-NTA column (HiTrap chelating HP) followed by gel filtration chromatography (HiLoad 16/60 superdex 200). Purified His-TEV-NQO1 was further cleaved by TEV protease and the His-TEV was removed upon binding to Ni-NTA column. For co-immunoprecipitation of endogenous SIRT and NQO1, mouse livers RIPA extracts were immunoprecipitated with either control protein A/G beads (Santa Cruz) or A/G beads coated with either non-immune rabbit IgG, or rabbit anti SIRT1 H300 (Santa Cruz). Protein levels were analyzed by Western blotting.
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