Anti-Hsp27 antibody [EPR5477] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (AB229442)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-HSPB1 knockout cells (red line) stained with ab109376. The cells were fixed with 4% formaldehyde (10 min)� and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab109376, 1�g/ml) for 30 min at 22�C. The secondary antibody used was Alexa Fluor^� 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22�C. A rabbit IgG isotype control antibody� (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-HSPB1 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109376).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (AB229442)

This ICC/IF data was generated using the same anti-Hsp27 antibody clone, EPR5477, in a different buffer formulation (cat# ab109376).

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling Hsp27 with purified ab109376 at 1/500. Cells were fixed with 100% methanol. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).

Secondary Only Control : PBS was used instead of the primary antibody as the negative control.

• WB

Lab

Western blot - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (AB229442)

This data was developed using the same antibody clone in a different buffer formulation (ab109376).

Lanes 1- 2 : Merged signal (red and green). Green - ab109376 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab109376 was shown to react with Hsp27 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261738 (knockout cell lysate ab256945) was used. Wild-type HeLa and HSPB1 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. ab109376 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 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-Hsp27 antibody [EPR5477] (<a href='/en-us/products/primary-antibodies/hsp27-antibody-epr5477-ab109376'>ab109376</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

HSPB1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human HSPB1 (Hsp27) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-hspb1-hsp27-knockout-hela-cell-line-ab261738'>ab261738</a>)

Predicted band size: 23 kDa

Observed band size: 23 kDa

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• WB

Lab

Western blot - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (AB229442)

This WB data was generated using the same anti-Hsp27 antibody clone, EPR5477, in a different buffer formulation (cat# ab109376).

Lanes 1 - 4 : Merged signal (red and green). Green - ab109376 observed at 27 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109376 was shown to specifically react with Hsp27 in wild-type cells as signal was lost in Hsp27 knockout HEP1 cells. Wild-type and Hsp27 knockout samples were subjected to SDS-PAGE. ab109376 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Hsp27 antibody [EPR5477] (<a href='/en-us/products/primary-antibodies/hsp27-antibody-epr5477-ab109376'>ab109376</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Hsp27 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Predicted band size: 23 kDa

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