western results

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jasmina
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western results

Post by jasmina » Thu Apr 08, 2010 9:12 am

hi there!
Today after detection, i got result as following (attachment). I think there are white bands which size is little bigger than 42KDa, and between each bands, i can see blackspot, i never got result like this before. confusing :?:
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100408 PP38111.jpg

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JackBean
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Post by JackBean » Thu Apr 08, 2010 9:30 am

what are the big black spots?
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Re:

Post by jasmina » Fri Apr 09, 2010 12:59 am

JackBean wrote:what are the big black spots?


I have no idea about it, previous, when i detected this antibody, expect aim bands, background is clear. so i do not know what happened this time. :?

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Re: western results

Post by jasmina » Fri Apr 09, 2010 6:29 am

I did detection again today, using same antibody, but varied protocol. yesterday, i used 5% BSA blocking, and blocked 1hr, then 1st antibody incubation overnight at 4 degree. today, i used 5% skim milk blocking, blocked overnight at 4 degree then incubated with 1st antibody 1hr at room temperature. result as following, this time, this no black spot. but I still do not know what difference for these 2 types blocking and incubation protocol. :?
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100409 pp38.jpg

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MrMistery
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Post by MrMistery » Sat Apr 10, 2010 3:18 am

how long do you usually block for? Maybe 1 hour block is just not enough? Though I must confess, I've never seen an anomaly like yours, I don't understand why BSA would preferentially bind to where the lanes are and not in between
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Post by jasmina » Sat Apr 10, 2010 5:59 am

MrMistery wrote:how long do you usually block for? Maybe 1 hour block is just not enough? Though I must confess, I've never seen an anomaly like yours, I don't understand why BSA would preferentially bind to where the lanes are and not in between


my antibody is phospho-p38 MAPK kinase(Thr180/Tyr182) antibody. Usually, for this antibody, we used 1hr 5% BSA blocking, and incubation overnigh with primary antibody. In recent months, we used same protocol but could not get clear bands, so i changed protocol.

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Post by MrMistery » Sun Apr 11, 2010 10:18 pm

maybe that antibody is from a bad stock or something. You could try to borrow some from someone else and see if you get the same result. But really, if the longer blotting works, probably sticking with that is the wisest choice.
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