Anti-FABP5 antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-FABP5 antibody (AB84028)

ICC/IF image of ab84028 stained HeLa cells. The cells were 4% PFA 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 (ab84028, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) 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 4% PFA fixed (10 min) Hek293, HepG2, MCF-7 cells at 5µg/ml, and in 100% Methanol fixed (5 min) HeLa, Hek293, HepG2, MCF-7 cells at 5µg/ml.

• IP

Lab

Immunoprecipitation - Anti-FABP5 antibody (AB84028)

FABP5 was immunoprecipitated using 0.5mg A431 whole cell extract, 5μg of Rabbit polyclonal to and 50μl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, A431 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40μl SDS loading buffer and incubated for 10min at 70°C; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab84028.

Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band : 16kDa; FABP5.

All lanes:

Immunoprecipitation - Anti-FABP5 antibody (ab84028)

Predicted band size: 15 kDa

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Exposure time: 20min

• WB

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Western blot - Anti-FABP5 antibody (AB84028)

Lanes 1-3 : Merged signal (red and green). Green - ab84028 observed at 17 kDa. Red - loading control ab8245 observed at 36 kDa.

ab84028 Anti-FABP5 antibody was shown to specifically react with FABP5 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265905 (knockout cell lysate ab257431) was used. Wild-type and FABP5 knockout samples were subjected to SDS-PAGE. ab84028 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 500 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-FABP5 antibody (ab84028) at 1/500 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

FABP5 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human FABP5 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-fabp5-knockout-hela-cell-line-ab265905'>ab265905</a>)

Lane 3:

PC-3 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: 15 kDa

Observed band size: 17 kDa

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• WB

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Western blot - Anti-FABP5 antibody (AB84028)

All lanes:

Western blot - Anti-FABP5 antibody (ab84028) at 1 µg/mL

Lane 1:

Human skin tissue lysate - total protein (<a href='/en-us/products/unavailable/human-skin-tissue-lysate-total-protein-ab30166'>ab30166</a>) at 10 µg

Lane 2:

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

Lane 3:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 4:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 15 kDa

Observed band size: 16 kDa

true

Exposure time: 3min