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Zaax's Content

There have been 3 items by Zaax (Search limited from 19-September 19)

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#10543 Tricine gel problem

Posted by Zaax on 31 January 2005 - 04:19 AM in Protein and Proteomics

Tricine coomasie stain

hi all

first question for me here.
please take a look at my image.

i made a standard tris-tricine gel in order to see expression of two small mol wight proteins that i hope i sucssesfully transfected into L8 cells.

this was my first tricine gel.

i then fixed it using gluteraldehyde and then stained with coomasie blue. yes i know coomasie is not the most sensitive method, i just feared i might have been losing my 7 and 9 KD proteins somehow in the transfere.

anyway it seems as if all the proteins are gettign stuck before reaching the 20 kd marker.

i might have added too much protein having loaded about 60 micrograms.. but the upper sizes seemed to resolve nicely. so i dont think thats the problem.

i used according to instructions, both a cothod and anode buffer etc...

i ran them for about 2.5 h @ 50-60 mAmp.

please any ideas? i wanted to see the expression of my protein domains.

by the end the buffer was slightly warm, but not hot...



#10705 Strange problem with actin control in Western blot

Posted by Zaax on 03 February 2005 - 04:19 AM in Protein and Proteomics

hmm, thats wierd.
i cant think of anything.

but from my experiance, i HATE striping membranes. i find a stripped membrane never works as well as a fresh one. maybe that was the problem, u may have stripped for too long or something.

But i do have an idea, to help you isolate the problem. Your protein is almost certainly larger than 75 kd, correct?

So, after transfer, before you wash away your ponsoue, use the red marking and cut your membrane just under the 75 or over the 60 Kd marker.

then you do not need to strip to blot for actin. just add to the lower membrane anti-actine and the other part, your antibody.


#8173 How to remove PCR inhibitors from DNA

Posted by Zaax on 25 October 2004 - 11:48 PM in Molecular Biology

abolishing? what do you mean?

if you are getting no result, and are using the qiagen kit, i dont see what can be inhibiting the reaction.

what are you doing the pcr ON? genomic dna? (try a touchdown pcr pogram) plasmid? (try lowering the annealing temp)

make sure your primers are good.

so many things can go wrong in pcr. but if you are using kit's i dont see how inhibiters is the answer.

but i might be wrong...

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