Anti-ROCK2 antibody [EPR7141(B)] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ROCK2 antibody [EPR7141(B)] - BSA and Azide free (AB238961)

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling ROCK2 with purified ab125025 at 1/250. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab7291 anti-Tubulin (mouse mAb) primary and ab150120 (AlexaFluor^®594 goat anti-mouse) secondary both at 1/1000 dilution. Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody and ab150120 (anti-mouse) secondary antibody were used. For negative control 2, ab7291 (mouse primary antibody) was used followed by ab150077 (anti-rabbit secondary antibody).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab125025).

• WB

Lab

Western blot - Anti-ROCK2 antibody [EPR7141(B)] - BSA and Azide free (AB238961)

This data was developed using the same antibody clone in a different buffer formulation (ab125025).

Lanes 1- 4 : Merged signal (red and green). Green - ab125025 observed at 175 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab125025 was shown to react with ROCK2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265679 (knockout cell lysate ab257643) was used. Wild-type HeLa and ROCK2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab125025 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 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-ROCK2 antibody [EPR7141(B)] (<a href='/en-us/products/primary-antibodies/rock2-antibody-epr7141b-ab125025'>ab125025</a>) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ROCK2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ROCK2 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-rock2-knockout-hela-cell-line-ab265679'>ab265679</a>)

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 161 kDa

Observed band size: 175 kDa

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• WB

Lab

Western blot - Anti-ROCK2 antibody [EPR7141(B)] - BSA and Azide free (AB238961)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : ROCK2 knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : A10 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab125025 observed at 165 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab125025 was shown to specifically react with ROCK2 when ROCK2 knockout samples were used. Wild-type and ROCK2 knockout samples were subjected to SDS-PAGE. ab125025 and ab8245 (loading control to GAPDH) were diluted 1/10 000 and 1/2000 respectively and incubated overnight at 4°C. 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 1/10,000 dilution for 1 h at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol and sodium azide (ab125025).

All lanes:

Western blot - Anti-ROCK2 antibody [EPR7141(B)] (<a href='/en-us/products/primary-antibodies/rock2-antibody-epr7141b-ab125025'>ab125025</a>)

Predicted band size: 161 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-ROCK2 antibody [EPR7141(B)] - BSA and Azide free (AB238961)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.