720070
antibody from Invitrogen Antibodies
Targeting: H2AC11
H2A.1b, H2A/p, H2AFP, HIST1H2AG, pH2A/f
Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [1]
- Immunocytochemistry [1]
- Flow cytometry [1]
- Chromatin Immunoprecipitation [1]
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Validation data
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- Product number
- 720070 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- H2AK5ac Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on nuclear enriched cell extracts (30 µg lysate) of HeLa (Lane1), HeLa treated with SAHA (0.5 uM/24 hrs) (Lane 2), HeLa treated with Sodium butyrate (5mM/24 hours) (Lane 3), HCT 116 (Lane 4), HCT 116 treated with SAHA (0.5 uM/24 hours) (Lane 5), HCT 116 treated with Sodium butyrate (5mM/24 hours) (Lane 6), Jurkat (Lane 7), Jurkat treated with SAHA (0.5 uM/24 hours) (Lane 8), Jurkat treated with Sodium butyrate (5mM/24 hours) (Lane 9) and K562 (Lane 10) and K562 treated with SAHA (0.5 uM/24 hrs) (Lane 11). The blot was probed with Anti-Histone H2AK5ac Rabbit Polyclonal Antibody (Product # 720070, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 diln). A clear 17kDa band corresponding to Histone H2AK5ac was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemilumines
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence was performed on fixed and permeabilized HeLa cells for detection of Histone H2AK5ac using Anti-Histone H2AK5ac Rabbit Polyclonal Antibody (Product # 720070, 0.5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:400). Panel a) shows representative cells that were stained for detection and localization of Histone H2AK5ac protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of Histone H2AK5ac. Panel e) represents control cells with no primary Antibody to assess background.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow Cytometry analysis of Histone H2AK5ac was performed on Jurkat cells labeled with Anti-Histone H2AK5ac Rabbit Polyclonal Antibody (Product# 720070, 2-4 ug/ 1M cells) or with Rabbit isotype control and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product # A27034, 0.4 ug/ml, 1:2500) as represented by the red and pink histograms respectively. The purple histogram represents unstained control cells and the green histogram represents no-primary-Antibody control. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (4468770).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Chromatin Immunoprecipitation (ChIP) was performed using Anti-Histone H2AK5ac Rabbit Polyclonal Antibody (Product # 720070, 5 µg) on sheared chromatin from 2 million HeLa cells treated with 5mM sodium butyrate for 24 hours using the "MAGnify ChIP system" kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Product # 4351106) with optimized PCR primer pairs for the promoters of the active cFOS region used as positive control target gene, and the region of the inactive SAT2 satellite repeat, used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.