CUT&Tag libraries were prepared using the Hyperactive in situ ChIP Library Prep Kit for Illumina (TD901, Vazyme Biotech, Nanjing, China). Briefly, cells were counted, and 100,000 cells were used for each assay. These cells were bound to the conA beads and incubated with anti-H3K27ac (ab4729, Abcam, Cambridge, UK) or anti-Nipbl antibody (A301-779A, Bethyl Laboratories, Montgomery, TX, USA) for 2 h at room temperature. After washing, cells were incubated with a secondary antibody (Goat Anti-Rabbit IgG, Sangon, Shanghai, China), and diluted (1:100) in the DIG Wash buffer for 1 h on a shaker with slow rotation at room temperature. The Hyperactive pG-Tn5 Transposase was then added, and cells were incubated for 1 h. After washing three times with the Dig-300 buffer, these cells were resuspended in the tagmentation buffer and incubated for 1 h at 37 °C. Chromatin was purified using a standard phenol/chloroform/alcohol extraction method. Fragmented DNA was then amplified by PCR and then purified by magnetic beads. The final libraries were sequenced on an Illumina platform.
Goat anti rabbit igg
Manufactured by Sangon
Sourced in China
Goat anti-rabbit IgG is a secondary antibody used for the detection and quantification of rabbit primary antibodies in various immunoassays and research applications. It is produced by immunizing goats with rabbit immunoglobulin G (IgG) and purifying the resulting antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Anti h3k27ac ab4729, by Abcam (1 mentions)
Rabbit anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Anti trpc4, by Alomone (1 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
Anti h3k4me3, by Diagenode (1 mentions)
Anti histone h2az, by Abcam (1 mentions)
Anti ctcf, by Abcam (1 mentions)
Goat anti mouse igg, by Sangon (1 mentions)
Anti h3k27ac, by Abcam (1 mentions)
Anti h3k27me3, by Abcam (1 mentions)
Trans blot, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
5 protocols using goat anti rabbit igg
1
CUT&Tag Chromatin Profiling
2
Western Blot Analysis of TRPC3 and TRPC4 Proteins
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Tissues or cells were homogenized and lysed in radioimmunoprecipitation assay buffer (Beyotime, Jiangsu, China), and then proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel before transferring onto nitrocellulose membrane. The blot was incubated with primary antibody (rabbit polyclonal anti-TRPC3 from Sangon, Shanghai, China, and anti-TRPC4 from Alomone Labs, Jerusalem, Israel) overnight at 4°C, washed with Tris-buffered saline and Tween 20, and then incubated with horseradish peroxidase−conjugated secondary antibody (goat anti-rabbit IgG, Sangon, Shanghai, China). Rabbit anti-GAPDH (anti-glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotech, USA) was used as an internal standard for protein quantification. Visualization was carried out using enhanced chemiluminescence detection reagents (Engreen, Beijing, China). Images were captured by a gel documentation system and the band density was analyzed using Quantity One (Bio-Rad, Hercules, USA).
3
CutAndTag Profiling of Placental Epigenome
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Digestion of placenta tissue follows the same protocol with bulk ATAC assay. After quantification, 50,000–100,000 cells were used for CutAndTag experiment. CutAndTag experiments were performed with NovoProtein CutAndTag 2.0 pAG-Tn5 kit (NovoProtein, #N259) according to the manufacturer’s protocol. Antibodies used in this study include the following: anti-H3K4me3 (Diagenode, #C154100003), anti-H3K27ac (Abcam, #ab4729), anti-CTCF (Abcam, #ab188408), anti-Histone H2A.Z (Abcam, #ab4174), anti-H3K27me3 (Abcam, Cat. #ab6002), goat anti-mouse IgG (Sangon, #D111024), and goat anti-rabbit IgG (Sangon, #D111018). Each library was sequenced to 2 × human genome coverage on Novaseq6000 sequencer (Illumina, CA) in PE150 format. Sequencing was performed at Berry Genomics, Beijing, China. QC metrics is described in Additional file 9 : Table S8.
4
SDS-PAGE and Immunoblot Analysis of Protein Expression
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Expression products in the medium, cell supernatant and pellet, purified proteins were determined by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The fresh medium was used as a negative control. After SDS-PAGE, the proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) using Trans-Blot (Bio-Rad, Hercules, CA, USA) at 20 V for 15 min. Immunoblot analysis was conducted, as described by Li, et al. [49 (link)]. Anti-BmSUC1 polyclonal antibody was used as the primary antibody (1:500) and goat anti-rabbit IgG as the secondary antibody (1:10,000) (Sangon, Shanghai, China). The final detection was performed by using Diaminobenzidine (DAB) Horseradish Peroxidase Color Development Kit (Sangon, Shanghai, China).
5
Immunohistochemical Evaluation of VCAN and THBS2
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Immunohistochemistry (IHC) was used to detect the protein expression of VCAN and THBS2. The high temperature and high pressure antigen retrieval method was used for antigen retrieval. The sections were separately added with Anti-THBS2 rabbit polyclonal antibody (Sangon Biotech Co., Ltd, Shanghai, China) or Anti-VCAN rabbit polyclonal antibody (Sangon Biotech Co., Ltd, Shanghai, China) and incubated in 2 hours. Then these sections were added with Goat anti-Rabbit IgG (Sangon Biotech Co., Ltd, Shanghai, China) and incubated in 20 min. Then Diaminobenzidine tetrahydrochloride (DAB) staining was used for staining. The immunohistochemical results were interpreted by two pathologists. THBS2 and VCAN protein expressions were evaluated by intensity of staining and percentage of stained tumor cells. Intensity was given scores 0–3 (0, no; 1, weak; 2, moderate; 3, strong) and the percentage of stained tumor cells in all tumor cells was given scores 0–4 (0:<5%, 1:5–25%, 2:>25–50%, 3:>50–75%, 4:>75%). The semiquantitative immunoreactivity scoring system (IRS) was used to score, that is the percentage fraction of stained tumor cells multiplied by the staining intensity fraction. The positive expression was defined as the score >3 and the negative expression was defined as the score ≤3.
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