Anti-Aryl hydrocarbon Receptor antibody [RPT1] (ab2770)

Thanks. It is great if you can send me a hard copy of these inserts..   Regards Shally  : 10/27/11  

ANSWER:

Thank you for your reply. I will be happy to mail the datasheets to you. Are you having trouble accessing the datasheets online? I only ask so that I can inform our IT department if there are problems. Could you also please send me the address that you would like the datasheets sent to. Thank you and I look forward to your reply.

Dear Technical, would you please mail us hard copy of thee inserts as we have confusion reading these files.  We hope to be able to receive them tomorrow if you send us today.  

ANSWER:

The datasheets are accessible from the product pages on the abcam.com website. You could try and print them from there. If you still have difficulty, please let me know and I will mail the datasheets to you.

Dear Technical, we do not have the product insert for the following items. would you please send us copy asap?   Best regard

ANSWER:

Thank you for contacting Abcam.

Please find attached the missing datasheets.

If there is anything else I can help you with, please let me kn

I am not seeing a signal in Western blotting. Can you tell me how the antibody was tested and what was used as a positive control?

ANSWER:

Thank you for your enquiry.

The antibody was tested on Hepa 1 cell lysate and according to Swiss-Prot ARH is expressed in all tissues tested, including blood, brain, heart, kidney, liver, lung, pancreas and skeletal muscle. I would suspect that any liver cell lysate should have ARH expression.

Can you fill out the form at the following link so I am better able to assist you?

http://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=2770&mode=questionaire

I look forward to hearing from you,

Customer is using this antibody in WB and is getting a very weak signal. Used the primary at 1:1000 and 1:500, and is using MCF7 cell lysate (loaded 100 ug).

What were the exact WB conditions that were used when this antibody was tested? Is the protocol available?

ANSWER:

Thank you for your phone call. The originator of ab2770 has provided the following suggestions as well as their general Western blotting protocol below. I hope this helps, if you need additional assistance, please do let me know.

1. The concentration of ab2779 may be insufficient. Use longer incubation times and higher concentrations of primary antibody. Be sure to run positive controls to ensure that the transfer system is working. How long are you incubating with ab2770? I would suggest incubating overnight at 4C.

2. Do not over wash the membrane as excessive washing can wash away the primary antibody.

3. 95 kDa is a fairly large size. Did all the protein elute from the gel? You can check this by using a coomassie stain on the gel to visualize and see if the high molecular weight proteins transfered.

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

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

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

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

5.Substrate incubation (ECL) a.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. b.Agitate bag or tray to cover the surface of the membrane. c.Remove the blot from the bag or tray and place it between two pieces of write-on transparency film. d.Smooth over the covered blot to remove air bubbles and excess substrate. e.Expose to X-ray film or any sensitive screen. (Check manufacturer’s instructions for specific ECL reagents and procedures.)