Anti-Endothelin 1 抗体 (ab117757)

Thank you for contacting us and your interest in our products.

I am sorry for the delay in getting back to you. I can confirm that the Anti-Endothelin 1 antibody (ab117757) has been used to perform IHC staining with paraffin embedded tissue with citrate buffer (pH 6) used for antigen retrieval and the antibody was incubated with the tissue overnight at 4°C at a dilution of 1/2000.

I hope this information has been helpful. Please do not hesitate to let me know if you need any further assistance.

Wenn wir keine genaueren Angaben zum Protokoll machen (wie in diesem Fall), bedeutet dies, dass Sie eigentlich ohne Probleme Ihr Standard-Protokoll verwenden können (Optimierungsschritte eingeschlossen).

Unfortunately, we do not have the blocking peptide for ab117757 available for sale. I apologize for saying it would be likely that we can make it available. If you cannot find an alternative blocking peptide source that overlaps the immunogen sequence for this antibody, below is a link to some commercial custom peptide manufacturers:
http://www.peptideresource.com/custom-peptide.html

Thank you for your inquiry and I am sorry to hear that you are having trouble with this antibody. I've copied our protocol below. Please letme know if this is similar to what you've tried.


Western Blot Protocol


Polyacrylamide gel electrophoresis and blottingAdd an appropriate amount of electrophoresis sample buffer (1X = 125mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, and 1% beta-mercaptoethanol) to all samples.
Heat to 95°C for 3-5 minutes.
Load 5-100 ug total protein in a volume that is appropriate for the size of the wells.
Electrophorese proteins for the appropriate time according to the manufacturers instructions.
Transfer proteins from the gel to a suitable membrane (e.g. nitrocellulose, PVDF, etc.) following the manufacturers protocol for transfer.
High molecular weight proteins (>120 kDa) can be wet transferred more efficiently if transfer time is increased and 0.05% SDS is included in the transfer buffer.


BlockingRemove the filter from the transfer apparatus and rinse in PBST/TBST to remove loose acrylamide.
Transferred proteins can be visualized by staining the membrane for 15-30 minutes with Ponceau S.
Remove stain from filter by washing with PBST/TBST.
Place filter into blocking solution.
Block for 30 minutes at 37°C, 1 hour at room temperature, or overnight at 4°C.


Incubation with primary antibodyDecant the blocking buffer and add the antibody, diluted in blocking buffer as suggested in the product description sheet.
Incubate with agitation for 30 minutes at 37°C, 1 hour at room temperature, or overnight at 4°C.


Incubation with secondary antibodyWash for 30 minutes with agitation in wash buffer (TBS or PBS with 0.1% Tween 20), changing the wash buffer every 5 minutes.
Decant the wash solution and add HRP-conjugated secondary antibody, diluted in 5% non-fat dry milk in wash buffer.
Incubate for 30 minutes at 37°C, 1 hour at room temperature, or overnight at 4°C.
Decant the antibody conjugate and wash for 30 minutes with agitation in wash buffer (TBS or PBS with 0.1% Tween 20), changing the wash buffer every 5 minutes.


Substrate incubation (ECL)Decant washing buffer and place the blot in a plastic bag or clean tray containing the development working solution (0.125 ml/cm2) for 1-5 minutes.
Agitate bag or tray to cover the surface of the membrane.
Remove the blot from the bag or tray and place it between two pieces of write-on transparency film.
Smooth over the covered blot to remove air bubbles and excess substrate.
Expose to X-ray film or any sensitive screen. (Check manufacturer's instructions for specific ECL reagents and procedures.)





I look forward to your reply.