Anti-Activin Receptor Type IA antibody (ab47086)

I will order one to try. Could you let me know what the dilution for

primary antibodies are?

ANSWER:

Thank you for your email. The antibody dilutions vary from antibody to antibody. For ab47086, the recommended dilution range is 3 - 5 µg/ml. For ab115301, the recommended dilution range is 0.3 - 1 µg/ml. But the optimal dilutions/concentrations should be determined by the end user. The WB dilutions can be found on the datasheet under Applications or Application Notes. If no concentration is given, a general starting dilution of 1 ug/ml is recommended for purified antibodies. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

for ab47086 and ab115301: WB protocol and any additional tips

ANSWER:

Thank you for contacting us. The WB protocol for ab47086 is as follows:

1. Equal amounts (about 10-50 μg/lane) of protein samples are resolved by SDS-PAGE and electro-blotted using a mini-gel transfer system onto a PVDF membrane using a current of 0.5-0.75 A for 1 hour. 2. The blots are stained with Amido black for 1 min and destained with 10% methanol plus 10% acetic acid. Amido black helps to monitor the efficiency of transfer without interfering with subsequent immunoreaction. Allow the blots to air dry until the color fades. 3. Wet the blots in 100% methanol, rinse with TBST, and then block for 1 hour with 5% Carnation nonfat dry milk in TBST (25 mM Tris-HCl, pH 8.0; 125 mM NaCl; 0.1% Tween 20). 4. The blots are incubated with primary antibody in 1% milk/TBST overnight at 4 degrees. The primary antibody should be used at the recommended concentration/dilution, and diluted with 1% Carnation nonfat dry milk in TBST. 5. After incubation with the primary antibody, the blots are washed five times in TBST and then incubated with the proper secondary antibody conjugated to horseradish peroxidase (HRP; according to manufacturer's instructions) for 60 minutes at RT. Dilute with 1% milk/TBST without sodium azide. 6. After five washes with TBST, the blots are developed for 5 minutes using Western Blot Chemiluminescent Substrate. 7. X-ray films are exposed to the blots for appropriate time periods. We normally expose the blots for 10 seconds, 1 minute, 5 minutes, and 20 minutes to visualize the chemiluminescence signal corresponding to the specific antibody-antigen reaction.

For ab115301, I will have to check with the lab regarding the WB protocol used. I will let you know when I hear back from them. Thank you for your patience. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information in the meantime.