Anti-KIF5A antibody

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KIF5A antibody (AB5628)

Immunofluorescence analysis of KIF5A in SH-SY5Y cells using ab5628. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton™ X-100, and blocked with 5% BSA. The cells were labeled with ab5628 at 1 µg/mL in 1% BSA and incubated for 3 hours at room temperature followed by a Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) Secondary Antibody at 1/2000 dilution for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with with DAPI. F-actin (Panel c : red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin at 1/300. (Panel d) represents the merged image showing cytoplasmic localization. (Panel e) shows the no primary antibody control. The images were captured at 60X magnification.

• IP

Supplier Data

Immunoprecipitation - Anti-KIF5A antibody (AB5628)

Immunoprecipitation of KIF5A in U-87 MG cells. Lysates were prepared and immunoprecipitation was performed using 3 μg of ab5628 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Immunoprecipitation - Anti-KIF5A antibody (ab5628) at 3 µg

All lanes:

U-87 MG cells

Observed band size: 117 kDa

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• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-KIF5A antibody (AB5628)

ICC/IF image of ab5628 stained PC12 cells. The cells were 4% formaldehyde 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 (ab5628, 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.

• WB

Lab

Western blot - Anti-KIF5A antibody (AB5628)

Western blot : Rabbit Polyclonal to KIF5A ab5628 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at kDa in Wild-type U-87 MG ab278079 cell lysates with no signal observed at this size in KIF5A knockout U-87 MG ab306717 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-KIF5A antibody (ab5628) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG ab278079 at 20 µg

Lane 2:

Western blot - Human KIF5A knockout U-87 MG cell line (<a href='/ko/products/cell-lines/human-kif5a-knockout-u-87-mg-cell-line-ab306717'>ab306717</a>) at 20 µg

Lane 3:

SK-N-FI at 20 µg

Lane 4:

HEK-293T at 20 µg

Secondary

All lanes:

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

Predicted band size: 117 kDa

Observed band size: 135 kDa

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• WB

Supplier Data

Western blot - Anti-KIF5A antibody (AB5628)

Blocking with 5 % skimmed milk

All lanes:

Western blot - Anti-KIF5A antibody (ab5628) at 2 µg/mL

Lane 1:

U-87 MG whole cell extracts at 30 µg

Lane 2:

HT-29 whole cell extracts at 30 µg

Lane 3:

C2C12 whole cell extracts at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG (H+L) HRP conjugate at 1/2500 dilution

Predicted band size: 117 kDa

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• WB

Supplier Data

Western blot - Anti-KIF5A antibody (AB5628)

ab5628 was shown to react with KIF5A in wild-type U-87 MG cells in Western blot with loss of signal observed in KIF5A knockout cell line ab306717. Wild-type U-87 MG and KIF5A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab5628 overnight at 4 °C at a 1/500 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-KIF5A antibody (ab5628) at 1/500 dilution

Lane 1:

Wild-type U-87 MG cells at 30 µg

Lane 2:

KIF5A knockout cell line at 30 µg

Lane 2:

Western blot - Human KIF5A knockout U-87 MG cell line (<a href='/ko/products/cell-lines/human-kif5a-knockout-u-87-mg-cell-line-ab306717'>ab306717</a>)

Predicted band size: 117 kDa

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• WB

CiteAb

Western blot - Anti-KIF5A antibody (AB5628)

KIF5A western blot using anti-KIF5A antibody ab5628. Publication image and figure legend from Kamata, H., Tsukasaki, Y., et al., 2017, Sci Rep, PubMed 28676645.

ab5628 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab5628 please see the product overview.

Upregulation of motor proteins during MesoMT. (A) mRNA level of motor proteins during MesoMT (n = 6). (B,C) Protein amount were investigated by western blot in both cell lysate (B) and culture medium (C). Full-length blots were presented in Supplementary Figs S2 and S3. (D) Quantitative analysis using densitometry of western blot of C (n = 3 for Col-1, n = 4 for PAI-1). *p < 0.05, **p < 0.01 vs basal level.

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