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Western blot detection other than antibody?


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5 replies to this topic

#1 pygo

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Posted 02 March 2005 - 12:26 AM

Hi guys,

Anyone know how to detect and quantify protein in western blot using method other than HRP-antibody? My lab is in tight budget and we are looking for cheaper alternative. Thanx heaps! B)

Sissle

#2 jadefalcon

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Posted 02 March 2005 - 04:06 AM

you can use alkaline-phosphatase (AP) coupled antibodies, but I'm not sure if this is cheaper than HRP, taken together the cost for the Ab and the substrate....

mike
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#3 margo33

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Posted 03 March 2005 - 01:54 AM

Je suis pas sûre que ça convienne mais c'est vraiment le moins coûteux: tu peux faire 1 coloration au rouge Ponceau ou ou bleu de Coomassie et regarder si tu as 1 bande à la taille que tu attends! Ensuite, si ce prétest est positif tu utilise les anticorps (moi aussi je prends des coumplés à la Phosphatase alcaline) pour confirmer la nature de ta protéine. Tu ne les utilises pas pour rien au moins. Bonne chance!

#4 jadefalcon

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Posted 03 March 2005 - 04:48 AM

  Je suis pas sûre que ça convienne mais c'est vraiment le moins coûteux: tu peux faire 1 coloration au rouge Ponceau ou ou bleu de Coomassie et regarder si tu as 1 bande à la taille que tu attends! Ensuite, si ce prétest est positif tu utilise les anticorps (moi aussi je prends des coumplés à la Phosphatase alcaline) pour confirmer la nature de ta protéine. Tu ne les utilises pas pour rien au moins. Bonne chance!


Errr, my french is a little bit rusty (i.e. plus mal), and there wasn't much french to begin with, so maybe people here can understand this way better than me, so corrections welcome!

I think margo33 says, you can stain your membrane with coommassie or ponceau and then use comparing methods to determine your protein-concentrations, besides she would use AP-antibodies, too, and wishes you luck!

(I hope this is halfway correct ^_^ )

mike

Edited by jadefalcon, 03 March 2005 - 04:49 AM.

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#5 margo33

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Posted 03 March 2005 - 05:20 AM

I wanted to say: you can stain your membrane with coommassie or ponceau and look if you've got a band at the size you 're waiting for. Then you can use AP-antibodies to confirm it's the right protein. So you don't waste your antibody for nothing!
Sorry for the answer in french!

#6 jadefalcon

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Posted 04 March 2005 - 06:04 AM

@margo:

I'd say: "swing and a miss", but a close one, sorry! :(

mike
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