Anti-acetyl Lysine抗体[1C6]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-acetyl Lysine antibody [1C6] (AB22550)

ICC/IF image of ab22550 stained MCF7 cells. The cells were 4% formaldehye fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab22550, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-acetyl Lysine antibody [1C6] (AB22550)

Immunoflouroescence analysis of HeLa Cells labelling lysine acetylated proteins with ab22550. Formalin fixed cells were permeabalized with 0.1& Triton X-100 in TBS for 10 mins at room temperature and subsequently blocked with BSA at room temperature for 15 mins. Cells were then probed with ab22550 at 1/100 for 1 hour at room temperature. The secondary used was a DyLight® 488 goat anti-mouse used at 1/400 for 30 minutes at room temperature. Additional counterstains used were F-actin with a DyLight® 554 Phalloidin and Neuclei stained using a Hoechst 33342 conjugate. Image was taken at X20 magnification.

• ChIP

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ChIP - Anti-acetyl Lysine antibody [1C6] (AB22550)

Chromatin Co-Immunoprecipitation (ChIP) analysis using ab22550 binding acetylated lysines in 10_^E+06 LNCaP cells. Protein binding was detected using real-time PCR.

Positive control : Fold enrichment of ab22550.
Negative Control : Non-specific IgG.

• WB

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Western blot - Anti-acetyl Lysine antibody [1C6] (AB22550)

Western blot analysis of lysine acetylated proteins from cells left untreated (DMSO only) or cells treated with 0.3uM or 3uM of Trichostatin A (TSA) for 16 hours was performed by loading 50 μg of the indicated whole cell lysates per well and 10 μL of PageRuler Prestained Protein Ladder onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with an Acetyl Lysine monoclonal antibody at a dilution of 1 : 1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1 : 20,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico.

All lanes:

Western blot - Anti-acetyl Lysine antibody [1C6] (ab22550) at 1/1000 dilution

Lane 1:

HeLa (untreated) cell lysate at 50 µg

Lane 2:

HeLa (treated with 0.3 uM TSA, 16h) cell lysate at 50 µg

Lane 3:

HeLa (treated with 3 uM TSA, 16h) cell lysate at 50 µg

Lane 4:

COS7 (untreated) cell lysate at 50 µg

Lane 5:

COS7 (treated with 0.3 uM TSA, 16h) cell lysate at 50 µg

Lane 6:

COS7 (treated with 3 uM TSA, 16h) cell lysate at 50 µg

Lane 7:

C2C12 (untreated) cell lysate at 50 µg

Lane 8:

C2C12 (treated with 0.3 uM TSA, 16h) cell lysate at 50 µg

Lane 9:

C2C12 (treated with 3 uM TSA, 16h) cell lysate at 50 µg

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