All lanes : Anti-RPS6 (phospho S240 + S244) antibody [EPR20770] (ab215214) at 1/5000 dilution

Lane 1 : MCF7 (human breast adenocarcinoma cell line) grown in serum-free media overnight, whole cell lysate
Lane 2 : MCF7 grown in serum-free media overnight, then grown in 20% FBS media for 30 minutes, whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast cell line) grown in serum-free media for 4 hours, whole cell lysate
Lane 4 : NIH/3T3 grown in serum-free media for 4 hours, then grown in 20% FBS media for 30 minutes, whole cell lysate
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma cell line) grown in serum-free media overnight, whole cell lysate
Lane 6 : PC-12 grown in serum-free media overnight, then grown in 20% FBS media for 30 minutes, whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 29 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

 

Exposure times: Lanes 1,2: 1 second; Lanes 3-6: 5 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast cell line) cells labeling RPS6 (phospho S240 + S244) with ab215214 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Cytoplasmic staining was increased after treatment with 20% FBS for 30 minutes on NIH/3T3 cells. Cells were FBS deprived for 4 hours before the treatment.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling RPS6 (phospho S240 + S244) with ab215214 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP), ready to use. Cytoplasmic staining on human placenta (image A), no signal was detected when tissues were treated with alkaline phosphatase (image B). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP), ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Dot blot analysis of RPS6 (phospho S240 + S244) labeled with ab215214 at 1/1000 dilution.

Lane 1: RPS6 (phospho S240 + S244) peptide.

Lane 2: RPS6 (phospho S240) peptide.

Lane 3: RPS6 (phospho S244) peptide.

Lane 4: RPS6 non-phospho peptide.

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

 

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast cell line) grown in serum-free media for 4 hours, then grown in 20% FBS media for 30 minutes (red) or NIH/3T3 grown in serum-free media for 4 hours (green), cell line labeling RPS6 (phospho S240 + S244) with ab215214 at 1/600 dilution compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/2000 dilution was used as the secondary antibody.

RPS9 (phospho S240 + S244) was immunoprecipitated from 0.35 mg of MCF7 (human breast adenocarcinoma cell line), grown in serum-free media overnight, then grown in 20% FBS media for 30 minutes, lysate with ab215214 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab215214 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Lane 1: MCF7 grown in serum-free media overnight, then grown in 20% FBS media for 30 minutes, whole cell lysate  10 μg (Input).

Lane 2: ab215214 IP in MCF7 grown in serum-free media overnight, then grown in 20% FBS media for 30 minutes, whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of  ab215214 in MCF7 grown in serum-free media overnight, then grown in 20% FBS media for 30 minutes, whole cell lysate.

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

Exposure time: 10 seconds.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling RPS6 (phospho S240 + S244) with ab215214 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP), ready to use. Cytoplasmic staining on human breast cancer (image A), no signal was detected when tissues were treated with alkaline phosphatase (image B). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP), ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling RPS6 (phospho S240 + S244) with ab215214 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP), ready to use. Cytoplasmic staining on mouse colon (image A), no signal was detected when tissues were treated with alkaline phosphatase (image B). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP), ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling RPS6 (phospho S240 + S244) with ab215214 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP), ready to use. Cytoplasmic staining on rat kidney (image A), no signal was detected when tissues were treated with alkaline phosphatase (image B). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP), ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.