Anti-RFP antibody [EPR18992]

3 product images

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  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) (Goat Anti-Rabbit IgG H&L (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 (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (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 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 at 1/1000 dilution.

  -ve control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 at 1/1000 dilution.

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  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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 26 kDa

  Observed band size: 26 kDa

  Exposure time: 3s

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  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] (Anti-alpha Tubulin antibody [DM1A] - Loading Control 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

  Performed under reducing conditions.

  Observed band size: 30 kDa