Recombinant Anti-ErbB4 / HER4 (phospho Y1284) antibody [EPR2273(2)] (ab109273)

Thank you for contacting us.

The lab has sent me the following protocol and information:

The ICC protocol used to test this antibody is attached. Cells are serum-starved for overnight, and then treated with 100 ng/ml EGF for 5 min at 37 deg C.

Immunofluorescent / Immunocytochemistry Protocol

1. Solutions and reagents

1.1. Washing buffer: PBST washing buffer: 1X PBS / 0.1% Tween-20 (Dulbecco’s Phosphate Buffered Saline)

1.2. 2% Paraformaldehyde (prepare fresh by dissolving paraformaldehyde in 1X PBS by heating at 70ºC until dissolving. Use cold.)

1.3. 0.1% Triton X-100

1.4. Blocking buffer: PBS (Dulbecco’s Phosphate Buffered Saline) + 10% serum (serum origin depends on the host of the secondary antibody)

1.5. Mounting medium for fluorescence.

1.6 Acetone (Production Grade)

2. Protocol

2.1. Grow cells in chamber slides or in 6-well tissue plates containing sterile coverslips. Prior to fixation, cells should not reach more than 80% confluency.

2.2. Fixation

2.2.1. Remove medium from chamber slides or 6-well plates. Wash once with PBS-T.

2.2.2. Fix cells with 2% paraformaldehyde for 20 min. For HeLa cells only, fix with acetone at -20 ºC for 5 mintues and continue to step 2.2.5.

2.2.3. Wash cells twice with 1XPBST.

2.2.4. Permeabilize cells with 0.1% Triton-X100 for 5 min

2.2.5. Wash cells twice with 1XPBST.

2.3. Blocking

2.3.1. Block cells with blocking buffer for 1 hour at room temperature (option - O/N 4ºC).

2.4. Staining

2.4.1. Dilute primary antibody in the blocking buffer per recommendation on the data sheet.

2.4.2. Apply primary antibody on the cells and incubate overnight at 4ºC.

2.4.3. Wash cells twice with 1X PBST.

2.4.4. Incubate cells in a dilution of fluorescently-labeled secondary antibody in PBS for 45 min at room temperature in the dark.

2.4.5. Wash cells three times with 1X PBST.

2.4.6. Counterstain cells with DAPI in a concentration of 300 nM in PBS for 5 min in the dark.

2.4.7. Wash cells three times with 1X PBST.

2.4.8. Mount slides with medium for fluorescent staining.

2.4.9. Store slides in the dark.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Thank you for your telephone call earlier today. According to Swissprot/Uniprot protein database site, the extracellular domain of the ErbB 4 is from amino acids 26 – 651. http://www.uniprot.org/uniprot/Q15303 We do have the following ErbB4 antibodies tested in ICC-If and human, but regrettably, their immunogen seuqences are all in the cytoplasmic domain. They have been tested in ICC on fixed cells. As discussed on the telephone, I am sorry they will not have been tested and guarnateed for live cell imaging. I am copying the details below in case they are still of interest to you. I can recommend to review the online datasheets for further information. ab19391 Anti-ErbB 4 antibody [HFR1] Mouse monoclonal Applications: Flow Cyt, ICC/IF, IHC-P, IP, WB Reacts with: Human, Mouse Immunogen: RSTLQHPDYLQEYST amino acids 1250-1264 Click here (or use the following: https://www.abcam.com/ErbB4--HER4-antibody-HFR-1-ab19391.html). ab109273 Anti-ErbB 4 (phospho Y1284) antibody [EPR2273(2)] Rabbit monoclonal Applications: Flow Cyt, ICC/IF, WB Reacts with: Human, Mouse, Rat Immunogen: residues surrounding phosphorylated Tyrosine 1284 Click here (or use the following: https://www.abcam.com/ErbB4--HER4-phospho-Y1284-antibody-EPR22732-ab109273.html). ab61059 Anti-ErbB 4 (phospho Y1284) antibody Rabbit polyclonal Applications: ELISA, ICC/IF, WB Reacts with: Human, Mouse, Rat Immunogen: phosphorylation site of tyrosine 1284 (P-E-YP-L-S) Click here (or use the following: https://www.abcam.com/ErbB4--HER4-phospho-Y1284-antibody-ab61059.html). ab63354 Anti-ErbB 4 antibody Rabbit polyclonal Applications: ELISA, ICC/IF, IHC-P, WB Reacts with: Human Immunogen: phosphorylation site of tyrosine 1284. Click here (or use the following: https://www.abcam.com/ErbB4--HER4-antibody-ab63354.html). I am sorry we do not have antibodies that exactly meet your requirements on this occasion, but I hope this will still be helpful. If you have any further questions, please do not hesitate to contact us.