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(無題) 削除/引用 No.4936-7 - 2016/03/22 (火) 08:47:39 - Tak Hi, Tokyo-san, Gotcha! today I run samples with 10% Page. This time I changed 1' Ab to anti-ActB. When next time also give same results, I will change 1'Ab and ECL. Best, Tak

(無題) 削除/引用 No.4936-6 - 2016/03/21 (月) 21:11:23 - TOKIO Hi, I think you should change the Ab. or ECL reagents. I recommend you to use anti-GAPDH from Cell Signaling and ECL Select from Amersham. They will improve your WB a lot. Good luck!

(無題) 削除/引用 No.4936-5 - 2016/03/21 (月) 14:33:24 - Tak Hi, Sorry I could not realize your name. Got it. I will test antibody against ActB tomorrow:9 Appreciated, Tak

(無題) 削除/引用 No.4936-4 - 2016/03/21 (月) 14:15:59 - おお > it not always guarantee the expression is easily detected. How easily expressions are detectable by WB also depends on antibody, so it is difficult to answer your question.

(無題) 削除/引用 No.4936-3 - 2016/03/21 (月) 13:55:28 - Tak Hi, thank for your reply. Got it. Actually I have been working on molecular biology for a ting time tho, I have not compared the expression differences among house-keeping proteins. My understanding of house-keeping proteins is that whose expression is pretty stable but it not always guarantee the expression is easily detected. I may rephrase that my question is which house-keeping proteins are most easy to detect. My western works well for another abundant proteins (exposure time was a couple of seconds). But my Gapdh this time was over 30 min, but the signal was pretty weak. but again, thanks for your helpful discussion and yeah, I keep these in mind. Cheers, Tak

(無題) 削除/引用 No.4936-2 - 2016/03/21 (月) 12:01:07 - おお Detecting Gapdh could not be difficult in general. Although I do not know how the Santa Cruz antibody works, you might want to have doubts about your samples, western blotting, etc as well as the antibody. Beta actin is one of the easiest proteins to detect by WB, so you can try if beta actin level does not change much among your samples. But keep what I mention above in mind.

For Western, what is the recommended house keeping proteins? 削除/引用 No.4936-1 - 2016/03/21 (月) 10:31:25 - Tak Hi, there, I wanted to ask you guys about which house keeping proteins can be easily detected by Western. I loaded 10 ug total proteins/lane in 10% Page and then blotted with rabbit anti-Gapdh pAb from Santa Cruz. But I faintly detected the band with 35 kDa. My sample is super-limited so I did not want to apply more samples per lane. So I am wondering if another house keeping protein(s) may be better in terms of more sensitivity or its abundances. Do you think anti-ActB or another proteins are better? My samples is total cell lysate from primary cancer cells and my purpose is to show equal loading among samples. Thank you so much for your help in advances, TaK

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