Total protein lysates were prepared in radioimmunoprecipitation assay buffer and quantified using the BCA Protein Assay (Thermo Fisher Scientific). Histones were extracted by an EpiSeeker histone extraction kit (Abcam). Protein was separated on 4 to 20% SDS–polyacrylamide gel electrophoresis gels, transferred to polyvinylidene difluoride membranes, and probed with antibodies against trimethylated histone H3, β-actin, p62, LC3 (Sigma-Aldrich), H3K4me3, H3K9me3 (Abcam), H3K27me3 (Millipore), H3K36me3, H3K79me3, histone H3, EZH2 (Cell Signaling), SETDB1 (Thermo Fisher Scientific), Atg5 (Cell Signaling), cyclophilin B (Abcam), and SUV39H1 (Santa Cruz Biotechnology). Quantification was performed using ImageJ after normalizing to loading controls.
Setdb1
Manufactured by Thermo Fisher Scientific
SETDB1 is a protein that functions as a histone methyltransferase, catalyzing the addition of methyl groups to lysine 9 of histone H3. This enzymatic activity is involved in the regulation of gene expression and chromatin structure.
Automatically generated - may contain errors
Lab products found in correlation
Paxillin, by Merck Group (2 mentions)
Vectashield mounting media, by Clinisciences (2 mentions)
Dmi6000, by Leica (2 mentions)
Phalloidin tritc, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lamin a c, by Merck Group (2 mentions)
E cadherin, by Abcam (2 mentions)
H3k27me3, by Diagenode (2 mentions)
H3k9me3, by Abcam (2 mentions)
H3k27me3, by Merck Group (1 mentions)
Episeeker histone extraction kit, by Abcam (1 mentions)
Suv39h1, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Cyclophilin b, by Abcam (1 mentions)
3 protocols using setdb1
1
Histone Modification and Autophagy Analysis
2
Immunofluorescence Staining of Histone Modifications
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Cells were seeded on glass coverslips at 60–70% confluence, fixed in 4% paraformaldehyde, incubated with 50 mM NH4Cl to quench formaldehyde, and permeabilized with 0.5% Triton X-100. Samples were subsequently incubated overnight with primary antibody against SETDB1 (Thermo Fisher; Cat#: MA5-15722), H3K9me3 (Abcam; Cat#: ab8898), H3K27me3 (Diagenode; Cat#: C15410069), Lamin A/C (Sigma-Aldrich; Cat#: SAB4200236), E-cadherin (Abcam; Cat#: ab40772), phalloidin-TRITC (Sigma-Aldrich; Cat#: P1951), paxillin (Millipore; Cat#: 05-417) overnight at 4°C, followed by incubation with appropriate fluorophore-conjugated secondary antibodies (Donkey F(ab’)2; Jackson ImmunoResearch Laboratories) for an additional 1 h. Cell nuclei were stained with DAPI (Life Technologies; Cat#: 62248). Coverslips were mounted with Vectashield mounting media (Clinisciences). Microscopy was performed using an inverted microscope Leica DMI-6000, using 40×, 63× or 100× immersion objectives. Images were taken with the HQ2 Coolsnap motorized by MetaMorph 7.10.2.240 software. All images were processed with ImageJ (Fiji) software.
3
Immunofluorescence Staining of Cell Lines
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Cells were seeded on glass coverslips at 60 -70 % confluence, fixed in 4 % paraformaldehyde, incubated with 50 mM NH4Cl to quench formaldehyde, and permeabilized with 0.5 % Triton X-100. Samples were subsequently incubated overnight with primary antibody against SETDB1 (Thermo Fisher; Cat#: MA5-15722), H3K9me3 (Abcam; Cat#: ab8898), H3K27me3 (Diagenode; Cat#: C15410069), Lamin A/C (Sigma-Aldrich; Cat#: SAB4200236), E-cadherin (Abcam; Cat#: ab40772), phalloidin-TRITC (Sigma-Aldrich; Cat#: P1951), paxillin (Millipore; Cat#: 05-417) overnight at 4 °C, followed by incubation with appropriate fluorophore-conjugated secondary antibodies (Donkey F(ab')2; Jackson ImmunoResearch Laboratories) for an additional 1 hour. Cell nuclei were stained with DAPI (Life Technologies; Cat#: 62248). Coverslips were mounted with Vectashield mounting media (Clinisciences). Microscopy was performed using an inverted microscope Leica DMI-6000, using 40x, 63x or 100x immersion objectives. Images were taken with the HQ2 Coolsnap motorized by MetaMorph 7.10.2.240 software. All images were processed with ImageJ (Fiji) software.
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