Jurkat whole cell lysate (ab7899)

Thank you for sending this data to me.

While reviewing this slide, I noted that your bands are white. Oversaturation of secondary antibodies can lead to masked signal or false positive. In this case, the secondary antibody which you are using at 1:2500, ab6721 has been used successful at dilutions at almost 100 times what you have used. This antibody can be diluted to 1:48,000 to 1:207,000 in many instances. I would recommend to titer this secondary antibody to find the optimal dilution for this product. As your bands are super saturated at 1:2500 I would recommend a titer range of ab6721 beginning at 1:10,000.

Hopefully by controlling the secondary antibody saturation, the true signal from these anti-PDGF antibodies can emerge.

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Thank you for contacting us.

I was not able to view the attachment included. Would you be able to re-send this to me as a jpeg, ppt or png file? Also could you let me know some of the details of the experiement (amount of positive control used, ab concentration,etc)?

I look forward to your reply. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Thank you for confirming these details and for your cooperation. I am sorry that you are continuing to have difficulties when using these products. As requested, I have issued a free of charge replacement of ab73229 PDGF BB Protein and have arrange for our Jurkat Cell lysate (ab7899) to be sent to you free of charge as well. The order number for these products is xxxx. To check the status of the order please contact our Customer Service team and reference this number. I do hope that by increasing the amount of this replacement protein to 100ng/well, by using the Jurkat lysate at 30ug/well a second control and by using an increased concentration of ab135881 to 1:500 as seen on the datasheet, you will be able to see good results. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

Thank you for contacting us.
According to the datasheet, the this protein is not expressed in most lymphoid cell lines: B-cells, T-cells and NK cells. Therefore, I would suggest to use a T-cell line like Jurkat lysate as a negative control.

https://www.abcam.com/index.html?datasheet=7899 https://www.abcam.com/index.html?datasheet=7899.

As a "negative control" for WB I woul suggest to perform a non-primary control, which simply means skipping the promary antibody incubation.

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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I'm sorry to hear you are experiencing problems with our Jurkat lysate ab7899 and have tried to look at the [ a competitor] antibodies to see if they were the same as ab12034 and ab12035. In fact they are not the same antibodies but the same clones, and the [ a competitor] antibodies come as "purified ascites" while the Abcam antibodies have been Protein G purified. It is likely that the purification process is therefore different and ascites will give more bands as it is not well purified. I would like to recommend asking [ a competitor] regarding that reported 125kda band with the antibodies, as this could be due to the purity of their products. I suspect the problem could also be due to the blocking agent in the experiment, especially if you see lots of non specific bands with your other lysates. We find that a number of antibodies bind non specifically if too much blocking agent is present, I would therefore recommend trying a 1hr incubation in milk 5% in TBST on a membrane and a one hour incubation in BSA 5% in TBST on another membrane. Then I recommend to wash lightly the membranes in TBST (10seconds) and incubate the primary antibody (and secondary) in TBST only. I hope these suggestions will help you, please do not hesitate to contact me if I can be of further help,

Please let me know what extra information you require. This product is a Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate which we sell in units of 100µg in 1 x SDS sample buffer containing 5% b-mercaptoethanol. There is further information about the cell line in the relevance section of the datasheet. I'm not sure what extra information you require.

Thank you for your email and I'm sorry to hear about the difficulty you are experiencing with ab4788. I apologize for the shortage that you received and can certainly send you a replacement vial free of charge. Can you please tell me the batch number that you received (it is located on the vial)? Also, what was the Abcam order number or purchase number that was used? Thank you, and I look forward to hearing from you.

Yes, Jurkat cell lysate would be a good positive control for active caspase 9. Jurkat cell lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl flouride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecylsulfate, 5 µg/ml of aprotinin, 5 µg/ml of leupeptin). Cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The cell lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol.