Anti-Chk2 (phospho T68) antibody

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Chk2 (phospho T68) antibody (AB85743)

ICC/IF image of ab85743 stained HepG2 cells. The cells were 4% formaldehyde 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 ab85743 at 5μg/ml overnight at +4°C. The secondary antibody (green) was goat anti rabbit DyLight® 488 IgG (H+L) (ab96899) used at a 1/1000 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. This antibody also gave a positive result in formaldehyde fixed (4%, 10min) HeLa cells at 5ug/ml.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 (phospho T68) antibody (AB85743)

IHC image of Chk2 staining in human colon adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab85743, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• WB

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Western blot - Anti-Chk2 (phospho T68) antibody (AB85743)

Secondary antibody - anti-rabbit HRP (IgG H&L; ab97080)

All lanes:

Western blot - Anti-Chk2 (phospho T68) antibody (ab85743) at 1 µg/mL

Lane 1:

HeLa Whole Cell Lysate - Irradiated (5Gy) at 10 µg

Lane 2:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

HeLa Whole Cell Lysate - Irradiated (5Gy) at 10 µg with Human Chk2 (phospho T68) peptide (<a href='/en-us/products/unavailable/human-chk2-phospho-t68-peptide-ab111650'>ab111650</a>)

Lane 4:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg with Human Chk2 (phospho T68) peptide (<a href='/en-us/products/unavailable/human-chk2-phospho-t68-peptide-ab111650'>ab111650</a>)

Lane 5:

HeLa Whole Cell Lysate - Irradiated (5Gy) at 10 µg with Human Chk2 peptide (<a href='/en-us/products/unavailable/human-chk2-peptide-ab118179'>ab118179</a>)

Lane 6:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg with Human Chk2 peptide (<a href='/en-us/products/unavailable/human-chk2-peptide-ab118179'>ab118179</a>)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 61 kDa

Observed band size: 145 kDa,38 kDa,70 kDa

true

Exposure time: 3min