Gonads of adult hermaphrodites (24 hours post L4) were dissected and stained as previously described [71 (link)]. Briefly, excised germlines were fixed for 10 minutes with 2% formaldehyde (Sigma Aldrich) in K2HPO4 (pH 7.2). Germlines were then freeze-cracked on dry ice and placed in cold methanol (Merck) for 5 minutes. Blocking was performed for 30 minutes in 1% BSA in PBST. Slides were incubated overnight at 4°C in a humid chamber with primary antibodies, washed 3 times for 10 minutes in PBST and incubated 2 hours with the secondary antibodies at room temperature. Slides were then washed 3 times for 10 minutes in PBST before being mounted on coverslips with Vectashield. In the second wash DAPI was added at a concentration of 100 ng/ml. Primary and secondary antibodies were diluted in blocking solution as follows: anti-RAD51 (Novus Biologicals; 29480002), 1:10000; anti-H3K36me2 (Active Motif; 61019) 1:200; donkey anti-mouse Alexa 488 (Life Technologies; A21202) 1:200, goat anti-rabbit Alexa 568 (Invitrogene; A11036) 1:200.
Anti h3k36me2
Manufactured by Active Motif
Sourced in United States
Anti-H3K36me2 is a laboratory product that detects the dimethylation of lysine 36 on histone H3 protein. This modification is associated with active transcription and is involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Ab2651, by Abcam (2 mentions)
Ab9050, by EpiCypher (2 mentions)
Anti h2b, by Active Motif (2 mentions)
Na934v, by GE Healthcare (2 mentions)
Anti g6pdh, by Merck Group (2 mentions)
Anti histone h3, by EpiCypher (2 mentions)
Anti flag m2 for flag tagged spt6, by Merck Group (2 mentions)
Anti histone h3k4me3, by EpiCypher (2 mentions)
Anti histone h3k79me3, by Abcam (2 mentions)
Anti histone h3k36me3, by Abcam (2 mentions)
Anti set2, by Active Motif (2 mentions)
Anti rnapii ser2p, by Active Motif (2 mentions)
Anti h2bk123ub1, by Cell Signaling Technology (2 mentions)
Anti mouse secondary, by GE Healthcare (2 mentions)
Hrp conjugated anti rabbit, by GE Healthcare (2 mentions)
Ecl prime, by Cytiva (2 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (2 mentions)
Anti h3k4me3, by Active Motif (2 mentions)
Anti h3k36me3, by Active Motif (2 mentions)
Alexa 568, by Thermo Fisher Scientific (1 mentions)
8 protocols using anti h3k36me2
1
Immunostaining of Hermaphrodite Gonads
2
Yeast Protein Extraction and Immunoblotting
3
ChIP-IT Express Protocol for Epigenetic Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ChIP-IT Express kit (Active Motif) was used for ChIP assay in this study following the manufacturer's protocol. The additional antibodies used in ChIP assay were rabbit IgG (Santa Cruz Biotechnology, sc-2027), anti-acetyl-histone H3 (Millipore, 06-599), anti-H3K36me2 (Active Motif, 39255), anti-H3K4me3 (Active Motif, 39159), and anti-H3K36me3 (Active Motif, 61101). These antibodies are all ChIP grade and have been validated by their companies. The ChIP PCR primers for amplification of ERα/ESR1 gene next to the promoter region (A) are: Forward, 5′-CCCACTCAACAGCGTGTCT-3′ Reverse, 5′-CTGCAGGAAAGGCGACAG-3′. The ChIP primers for amplification of ERα gene in the coding region (B) are: Forward, 5′-GAAGAAGCATGGGTAAATGTCA-3′ Reverse, 5′-TCAGCCCTGAACCCAGTG-3′.
4
Chromatin Immunoprecipitation of H3K36me2 and NSD2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Control and NSD2-KD1 143B cells (2 × 107 cells) were crosslinked, lysed and sheared using a UCD-300 (Bioruptor) to ~200–700 base pairs in length. ChIP was then performed using an EZ ChIP Kit (Millipore; 17-371) according to the manufacturer’s instructions. ChIP-enriched DNA was then quantified by RT-qPCR. The ChIP antibodies used were anti-H3K36me2 (Active Motif) and anti-NSD2 (Abcam). The primer sequences are listed in Table S3 .
5
Chromatin Immunoprecipitation Profiling Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following commercially available antibodies were used:
Anti-H3 (1:5.000, #61475), Anti-H3K36me3 (1:4.000, #61102), Anti-H3K36me2 (1:1.000, #39255), Anti-H3K36me1 (1:1.000, #61351), Anti-H3K4me3 (1:500, #61379) and anti-H2A (1:1.000, #39209) were from Active Motif. Anti-H3K27me3 (1:1.000, C15410069), Anti-H3K79me3 (1:1.000, C15310068), anti-H2A.Zac (2 µg/IP, C15410202-050) and Anti-H3K9me3 (1:1.000, C15410056) were from Diagenode. Anti-H4K20me3 (1:1.000, ab9053), Anti-H3 (1:30.000, ab1791), anti-MTA1 (1:1.000, ab71153 or 3 µg/IP, ab50209), anti-CHD4 (1:1.000 or 2 µg/IP, ab70469), anti-HDAC2 (1:5.000, ab124974) anti-RBBP4 (1:1.000, ab488), anti-RBBP7 (1:1.000, ab3535), Anti-H3K27ac (2 µg/IP, ab4729), anti-H2A.Z (1:3.000, ab4174), and anti-MBD3 (1:5.000, ab157464) were from Abcam. Anti-PWWP2A (1:1.000, NBP2-13833) from Novus (Acris). Anti-HA-HRP (1:40.000, 2999S) and anti-HDAC1 (1:20.000, 5356S) from Cell Signaling Technology. Anti-FLAG M2 (1:80.000 or 3 µg/IP, F1804) and anti-mouse IgG (3 µg/IP, I8765) from Sigma-Aldrich.
The following secondary antibodies were used:
Anti-rabbit IRDye 800CW (1:10.000, 926-32211) and anti-mouse IRDye 680RD (1:10.000 926‐68070) from LI-COR Biosciences. Anti-mouse-HRP (1:10.000, M114) from Leinco Technologies. Anti-mouse and anti-rabbit HRP-linked antibodies (1:10.000, NA931 and NA934) from VWR.
Anti-H3 (1:5.000, #61475), Anti-H3K36me3 (1:4.000, #61102), Anti-H3K36me2 (1:1.000, #39255), Anti-H3K36me1 (1:1.000, #61351), Anti-H3K4me3 (1:500, #61379) and anti-H2A (1:1.000, #39209) were from Active Motif. Anti-H3K27me3 (1:1.000, C15410069), Anti-H3K79me3 (1:1.000, C15310068), anti-H2A.Zac (2 µg/IP, C15410202-050) and Anti-H3K9me3 (1:1.000, C15410056) were from Diagenode. Anti-H4K20me3 (1:1.000, ab9053), Anti-H3 (1:30.000, ab1791), anti-MTA1 (1:1.000, ab71153 or 3 µg/IP, ab50209), anti-CHD4 (1:1.000 or 2 µg/IP, ab70469), anti-HDAC2 (1:5.000, ab124974) anti-RBBP4 (1:1.000, ab488), anti-RBBP7 (1:1.000, ab3535), Anti-H3K27ac (2 µg/IP, ab4729), anti-H2A.Z (1:3.000, ab4174), and anti-MBD3 (1:5.000, ab157464) were from Abcam. Anti-PWWP2A (1:1.000, NBP2-13833) from Novus (Acris). Anti-HA-HRP (1:40.000, 2999S) and anti-HDAC1 (1:20.000, 5356S) from Cell Signaling Technology. Anti-FLAG M2 (1:80.000 or 3 µg/IP, F1804) and anti-mouse IgG (3 µg/IP, I8765) from Sigma-Aldrich.
The following secondary antibodies were used:
Anti-rabbit IRDye 800CW (1:10.000, 926-32211) and anti-mouse IRDye 680RD (1:10.000 926‐68070) from LI-COR Biosciences. Anti-mouse-HRP (1:10.000, M114) from Leinco Technologies. Anti-mouse and anti-rabbit HRP-linked antibodies (1:10.000, NA931 and NA934) from VWR.
6
Yeast Protein Extraction and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yeast strains of the indicated genotypes (and their wild-type counterparts) were grown in YPD either at permissive or restrictive temperatures. Overnight-saturated cultures were diluted to an OD600 of 0.2 and allowed to grow until they reached an OD600 of 1. Five OD600 equivalents of cells were lysed using a modified TCA extraction method as described (Keogh et al., 2006a (link), 2006b (link)). 10–20 mg of the lysates were separated by SDS-PAGE and variously probed with the following antibodies: anti-FLAG-M2 [for FLAG tagged Spt6] (Sigma-Aldrich, F1804; 1:5000), anti-G6PDH (Sigma-Aldrich, A9521; 1:100,000), anti-histone H3K4me3 (EpiCypher, 13–0004; 1:5000), anti-histone H3K79me3 (Abcam, ab2651, 1:2500) anti-histone H3K36me3 (Abcam, ab9050, ab9050; 1:1000), anti-histone H3 (EpiCypher, 13–0001; 1:50,000), anti-Spt16 (gift from Tim Formosa University of Utah, 1:5000), anti-H3K36me2 (Active Motif, 39255; 1:1000), anti-Set2 (Generated in the Strahl lab, 1:5000), anti-RNAPII-Ser2P (Active Motif, Clone #3E10, 61084; 1:100), anti-H2BK123ub1 (Cell Signaling Technology, 5546; 1:2000), and anti-H2B (Active Motif, 39237; 1:2000). HRP-conjugated anti-rabbit (GE Healthcare, NA934V; 1:10,000) and anti-mouse secondary (GE Healthcare, NA931V; 1:10,000), antibodies were used at 1:1000 and proteins were detected using ECL Prime or enhanced chemiluminescence ECL (Amersham Biosciences).
7
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted under the indicated conditions, and the protein lysate was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The proteins were then transferred to polyvinylidene fluoride membranes. After being blocked in 10% non-fat milk, the membranes were incubated with specific primary and secondary antibodies. The antibodies used were anti-SOX2 (Active Motif), anti-NSD2 (Cell Signaling Technology, CST), anti-H3K36me2 (Active Motif), anti-H3K27me3 (CST), anti-BCL2 (CST), anti-GAPDH (CST), anti-H3 (Abways), anti-ERK (CST), anti-pERK (CST), anti-AKT (CST) and anti-pAKT (CST).
8
Xenograft Model of Salivary Adenoid Cystic Carcinoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Xenograft mouse models of salivary adenoid cystic carcinoma were established by subcutaneously injecting 1×10 6 transfected SACC cells in the flank of nude mice. Tumor growth was measured weekly with calipers. Tumor volume was calculated using the formula (L × W 2 ) × ½, where W and L represent the perpendicular minor and major dimension, respectively. All animals were sacrificed 21 days after tumor cell implantation. At autopsy the tumor was removed and weighed.
ChIP-qPCR analysis. 2×10 7 SACC-83 and WHSC1-knockdown SACC-83 cells were crosslinked, lysed and sheared to about 200-700 DNA base pairs in length using UCD-300 (Bioruptor, BE). ChIP was performed using Magnetic ChIP kit according to manufacturer's instructions Millipore) . Quantification of ChIP-enriched DNA was then performed by qPCR. The ChIP antibodies used were anti-H3K36me2 (61019, Active Motif, CΑ, USA) and normal rabbit IgG. Primers used are listed in Table ΙΙ.
ChIP-qPCR analysis. 2×10 7 SACC-83 and WHSC1-knockdown SACC-83 cells were crosslinked, lysed and sheared to about 200-700 DNA base pairs in length using UCD-300 (Bioruptor, BE). ChIP was performed using Magnetic ChIP kit according to manufacturer's instructions Millipore) . Quantification of ChIP-enriched DNA was then performed by qPCR. The ChIP antibodies used were anti-H3K36me2 (61019, Active Motif, CΑ, USA) and normal rabbit IgG. Primers used are listed in Table ΙΙ.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!