Anti-CNOT7 antibody [EPR18722]

• IP

Supplier Data

Immunoprecipitation - Anti-CNOT7 antibody [EPR18722] (AB195587)

CNOT7 was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab195587 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab195587 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate 10μg (Input).

Lane 2 : ab195587 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab195587 in HeLa whole cell lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-CNOT7 antibody [EPR18722] (ab195587)

Predicted band size: 33 kDa

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• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CNOT7 antibody [EPR18722] (AB195587)

Intracellular Flow Cytometry analysis of 4% paraformaldehyde fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cell linelabelling CNOT7 with ab195587 at 1/1000 dilution (red), isotype control with ab172730 (black) and unlabelled control cells without incubation with primary antibody and secondary antibody (blue).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CNOT7 antibody [EPR18722] (AB195587)

Intracellular Flow Cytometry analysis of 4% paraformaldehyde fixed F9 (Mouse embryonic testicular cancer cell line) cells labeling CNOT7 with ab195587 at 1/1000 dilution (red), isotype control with ab172730 (black) and unlabeled control cells without incubation with primary antibody and secondary antibody (blue).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CNOT7 antibody [EPR18722] (AB195587)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized F9 (Mouse embryonic testicular cancer cell line) cells labeling CNOT7 with ab195587 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows :
-ve control 1 : ab195587at 1/1000 dilution followed by Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary at 1/XXXX dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CNOT7 antibody [EPR18722] (AB195587)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling CNOT7 with ab195587 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows :
-ve control 1 : ab195587at 1/1000 dilution, followed by Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

• WB

Supplier Data

Western blot - Anti-CNOT7 antibody [EPR18722] (AB195587)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CNOT7 antibody [EPR18722] (ab195587) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human fetal spleen lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

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Exposure time: 30s

• WB

Lab

Western blot - Anti-CNOT7 antibody [EPR18722] (AB195587)

Lanes 1-3 : Merged signal (red and green). Green - ab195587 observed at 33 kDa. Red - loading control ab7291 observed at 50 kDa.

ab195587 Anti-CNOT7 antibody [EPR18722] was shown to specifically react with CNOT7 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265811 (knockout cell lysate ab258370) was used. Wild-type and CNOT7 knockout samples were subjected to SDS-PAGE. ab195587 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CNOT7 antibody [EPR18722] (ab195587) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CNOT7 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CNOT7 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cnot7-knockout-hela-cell-line-ab265811'>ab265811</a>)

Lane 3:

MOLT-4 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

• WB

Supplier Data

Western blot - Anti-CNOT7 antibody [EPR18722] (AB195587)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 1 seconds; Lane 2-4 : 2 seconds.

All lanes:

Western blot - Anti-CNOT7 antibody [EPR18722] (ab195587) at 1/1000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 10 µg

Lane 3:

NTERA-2/D1 (Human malignant pluripotent embryonic carcinoma cell line) whole cell lysate at 10 µg

Lane 4:

NCCIT (Human pluripotent embryonic carcinoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

• WB

Supplier Data

Western blot - Anti-CNOT7 antibody [EPR18722] (AB195587)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 2 seconds; Lane 2-6 : 5 seconds.

All lanes:

Western blot - Anti-CNOT7 antibody [EPR18722] (ab195587) at 1/1000 dilution

Lane 1:

Mouse ovary lysate at 10 µg

Lane 2:

Mouse embryo lysate at 10 µg

Lane 3:

F9 (Mouse embryonic testicular cancer cell line) whole cell lysate at 10 µg

Lane 4:

SP2/0 (Mouse spleen cell line) whole cell lysate at 10 µg

Lane 5:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 6:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

• WB

Supplier Data

Western blot - Anti-CNOT7 antibody [EPR18722] (AB195587)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2 : 15 seconds; Lane 3/4 : 5 seconds; Lane 5 : 30 seconds.

All lanes:

Western blot - Anti-CNOT7 antibody [EPR18722] (ab195587) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Rat kidney lysate at 10 µg

Lane 4:

Rat spleen lysate at 10 µg

Lane 5:

Rat heart lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

false