Anti-KMT6 / EZH2 antibody [SP129] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KMT6 / EZH2 antibody [SP129] - BSA and Azide free (AB245738)

Formalin-fixed, paraffin-embedded human prostate cancer tissue stained for KMT6 / EZH2 using ab227648 at 1/100 dilution in immunohistochemical analysis.

This data was developed using the same antibody clone in a different buffer formulation containing PBS#44; BSA#44; and sodium azide (ab227648)

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-KMT6 / EZH2 antibody [SP129] - BSA and Azide free (AB245738)

Flow cytometric analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling KMT6 / EZH2 with ab227648 at 1/100 dilution (green) compared with a negative control of rabbit IgG (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (ab227648).

• IP

Lab

Immunoprecipitation - Anti-KMT6 / EZH2 antibody [SP129] - BSA and Azide free (AB245738)

This data was developed using ab227648, the same antibody clone in a different buffer formulation.

KMT6 / EZH2 was immunoprecipitated from 0.35mg lysate HEK-293 (human embryonic kidney epithelial cell) whole cell lysate with ab227648 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab227648 at 1/1000 dilution. Capture antibody, 1 : 30 dilution (2μg in 0.35mg lysates) VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible.

Rabbit monoclonal IgG (ab172730) Isotype Control was used instead of ab185971 in HEK-293 whole cell lysate (Lane 3).

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible.

All lanes:

Immunoprecipitation - Anti-KMT6 / EZH2 antibody [SP129] (<a href='/en-us/products/primary-antibodies/kmt6-ezh2-antibody-sp129-ab227648'>ab227648</a>) at 1/30 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate 10 μg

Lane 2:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 85 kDa

false

Exposure time: 32s

• WB

Lab

Western blot - Anti-KMT6 / EZH2 antibody [SP129] - BSA and Azide free (AB245738)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227648).

Western blot : Anti-KMT6 / EZH2 antibody [SP129] ab227648 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 85 kDa in Wild-type A375 cell lysates with no signal observed at this size in EZH2 knockout A375 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-KMT6 / EZH2 antibody [SP129] (<a href='/en-us/products/primary-antibodies/kmt6-ezh2-antibody-sp129-ab227648'>ab227648</a>) at 1/1000 dilution

Lane 1:

Wild-type A375 cell lysates at 20 µg

Lane 2:

EZH2 knockout A375 cell lysates at 20 µg

Lane 3:

HEK-293 cell lysates at 20 µg

Lane 4:

MCF7 cell lysates at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 85 kDa

Observed band size: 85 kDa

false

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-KMT6 / EZH2 antibody [SP129] - BSA and Azide free (AB245738)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 K-562 (Human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab227648 [SP129]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (ab227648).