Anti-RFP antibody [EPR18992]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RFP antibody [EPR18992] (AB185921)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Empty vector transfected or RFP transfected HEK-293 (Human epithelial cell line from embryonic kidney) cells labeling RFP with ab185921 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing positive staining after transfection of RFP on HEK-293 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR18992] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab185921 at 1/100 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• WB

Lab

Western blot - Anti-RFP antibody [EPR18992] (AB185921)

Western blot : Anti-RFP antibody [EPR18992] (ab185921) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab185921 was shown to bind specifically to RFP. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-RFP antibody [EPR18992] (ab185921) at 1/1000 dilution

Lane 1:

HEK-293T mock transfected cell lysate at 20 µg

Lane 2:

HEK293 RFP transfected cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 30 kDa

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• WB

Lab

Western blot - Anti-RFP antibody [EPR18992] (AB185921)

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

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade - Used as Loading Control (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-RFP antibody [EPR18992] (ab185921) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a His tag whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a full-length RFP （eqFP611， Uniprot: Q8ISF8）expression vector containing a His-tag whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a full-length mRuby expression vector containing a His-tag whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a full-length mCherry (Uniprot: X5DSL3） expression vector containing a His-tag whole cell lysate at 20 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 1/20000 dilution

Observed band size: 26 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-RFP antibody [EPR18992] (AB185921)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-RFP antibody [EPR18992] (ab185921) at 1/10000 dilution

Lane 1:

RFP transfected HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 2:

Empty vector (vector control) transfected HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 3:

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

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg

Lane 5:

Human kidney lysate at 10 µg

Lane 6:

Human liver lysate at 10 µg

Lane 7:

Mouse brain lysate at 10 µg

Lane 8:

Mouse heart lysate at 10 µg

Lane 9:

Rat brain lysate at 10 µg

Lane 10:

Rat heart lysate at 10 µg

Lane 11:

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

Lane 12:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 13:

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

Lane 14:

NIH/3T3 (Mouse embryonic fibroblast 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: 26 kDa

Observed band size: 26 kDa

false

Exposure time: 3s