Anti-PLK4 抗体 (ab2642)

I have organised a new vial to be sent to you, you should get a confirmation e-mail for this "order" soon. Please could you let me know how you get on with this batch? I would very much appreciate your feedback.

The antibody in stock is a new batch and therefore has not been tested by other customers. I have arranged a credit note to be issued to you. This antibody sells well and the reported problems are therefore few. I wish you all the best with your research,

I'm terribly sorry you are having problems with ab2642. We have a new batch in stock which I could send to you or I can arrange a refund or credit note. Please let me know which option you would prefer and accept my apologies for this problem, I look forward to hearing from you,

Thank you for your e-mail. I am very sorry you are not getting the right band, especially with your positive control. I will arrange for a new replacement vial to be sent to you. I understand you bought the antibody from Gene Research. Can you please confirm this and I will straight away arrange with them to send you a new vial. I look forward to hearing from you soon,

Thank you for the information. I have now a clearer idea of your problem and would like to help by suggesting some modifications to your protocol. I think the problem might be a degradation of your protein before it is recognised by the antibody as you mention freeze thawing it several times and I would like to recommend another lysis buffer (RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton -100, 1% Sodium deoxycholate, 0.1% SDS). Following homogenisation over 20 minutes on ice centrifuge at 13,000 rpm for 5 min in a centrifuge (which is pre-cooled and stays cool)transfer the supernatant into clean tubes and do the protein concentration as usual. Add an equal volume of 2 x SDS sample buffer (made with SDS and betamercaptoethanol) and boil for 5 minutes. You can then store the lysates at -80C until use. I think the rest of your WB protocol looks fine. To minimise the background you report I would suggest blocking your membrane in non-fat milk (3-5%w/v in TBST, filtered) for one hour and not incubating the other antibodies in blocking buffer (just TBST). After blocking, rinse once (for 10sec)the membrane with TBST.This will remove excess milk which can cause sometimes high background. It could also be due to too much secondary antibody or simply that the secondary has gone off...have you tried incubating with no primary, this will help determining if the problem comes from the secondary. Try also the secondary in other WBs for the proteins and see if it gives you problems. Also, wash extensively the membrane between steps, it's always better to wash many times for short periods of time (4-5min) than a few long washes. I hope this information helps, please do not hesitate to contact me again if you still have problems, Good luck!

Thank you for your e-mail. Please find enclosed the protocol. Tissue Lysis: Tissue chunks were weighed and cut into approx 1mm cubes using a razor blade. The tissue was transferred to a handheld homogenizer and 3 ml of ice-cold RIPA buffer was added per 1g of tissue. The tissue was gently homogenised over 20 minutes on ice. The resulting lysate was aliquotted into 1.5 ml microfuge tubes and centrifuged at 13,000 rpm for 5 min in a microfuge. The supernatant was transferred into clean tubes and its protein concentration was measured with BioRad protein assay. The concentration was then adjusted to 5 mg/ml with RIPA lysis buffer. An equal volume of 2 x SDS sample buffer was added and the cell lysate was boiled for 5 minutes. Lysates were stored at -80C until use.(RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton -100, 1% Sodium deoxycholate, 0.1% SDS). We hope this will help.

Thank you for your enquiry and for forwarding your detailed protocol. This antibody has been tested for Western blot application using Human Placenta Lysate. We would strongly suggest running a positive control along with the samples. Also you need to run "no primary" control to see if the secondary antibody gives a clear background. You have mentioned in your e-mail that "Blocked by drying in fume hood overnight" - we are very sorry but haven't heard of anyone doing this before. You may wish to try the following: after protein transfer on PVDF membrane, block the membrane in 2.5% skimmed milk in TBS-T for 1 hr at room temperature with agitation. Note that if PVDF membranes dry out after transfer therefore they should be "rewetted" with 100% methanol – there should be some information about this in the membrane product literature. We hope this will help. Should you still need more technical support, please do not hesitate to contact us again.