Application
Immunoprecipitation
Sample
Human Cell lysate - nuclear (Human Embryonic Kidney (HEK293T))
Total protein in input
500 µg
Immuno-precipitation step
Other - Protein G Magnetic Beads
Specification
Human Embryonic Kidney (HEK293T)
Other product details
Incubation time
14 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: Nuclear Lysis Buffer
Concentration
4 µg/mg lysate
Western Blot antibody
Dilution
1/1000
Name
Abcam antibody:Anti-Histone H3 (tri methyl K36) antibody - ChIP Grade
Additional data
Additional Notes
IP Specificity Test: H3K36me3 (ab9050) antibody tested for IP. 500ug or 750ug total protein was used for IP with 2ug H3K36me3 antibody (ab9050). Briefly, antibody was incubated with lysate from human embryonic kidney HEK293T cells or mouse embryonic stem cells for 6 hours with rotation at 4C (Lysis buffer: 10mM Tris-HCl pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, protease inhibitors). Antibody+protein was then incubated overnight with 30uL Invitrogen Protein G Dynabeads (100003D) at 4C. Beads were was 5x in lysis buffer. IP’ed protein and antibody was eluted by boiling beads in 25uL 1X NuPAGE LDS Sample Buffer (NP0007) + 1mM DTT for 10 minutes. Samples were loaded onto a NuPAGE™ Novex™ 4-12% Bis-Tris protein gel (NP0321PK2). Bio-Rad Precision Plus Protein™ Dual Color Standard (#1610374) loaded for size reference (lane 1). Gel run in 1X NuPAGE MOPS SDS Running Buffer (NP0001) at room temperature and then transferred to a methanol-activated Novex™ PVDF membrane (LC2002) using a Invitrogen Mini Blot Module (B1000) in 1X NuPAGE Transfer Buffer (NP0006) at 20V for 2 hours at room temperature. Membrane was rinsed in 1X TBS-T and incubated in blocking solution (5% milk in 1X TBS-T) for 1 hour at room temperature on a rocking platform. H3K36me3 detection was performed by incubating blot with rabbit polyclonal antibody (ab9050 1mg/mL) diluted at 1:1000 in blocking solution for 1 hour at room temperature on a rocking platform. Primary antibody was washed 10 times with 1X TBS-T over the course of 1 hour at room temperature on a rocking platform. Secondary antibody binding was performed by incubation with Cell Signaling goat anti-rabbit IgG-HRP secondary (7074S) diluted at 1:5000 in blocking solution for 45 minutes at room temperature on a rocking platform. Following washes (above), blot was incubated for 2 minutes in Amersham ECL Prime (RPN2236) and chemiluminescent imaging was performed on a Fujifilm Imager (LAS-3000) on autoexposure.
Conclusions: H3K36me3 antibody works for western blot, but failed to IP the band detected in input sample. Only heavy and light chain antibody bands are visible in all three IP samples.
Abcam response
We would like to thank the reviewer for submitting this Abreview for this product. As the product had not yet been tested in this application on this tissue type we were unsure what the result might be and we appreciate the time and effort that the reviewer has put in to testing this product in this application. The customer has received value off a future purchase for performing these tests. We wish to thank them for participating in our AbTrial program.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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提交于 Sep 14 2017