How much starting material for an IP? - (May/09/2008 )

Hi all,

I'd like to do an IP to see if my tagged protein interacts directly with another protein, just via western, but I'm not sure how much material I'll need to start with. I'll be doing it on a particular dissected tissue from drosophila ovaries, only about 50um long, so I'd like to know roughly how much to dissect. I'm expecting the proteins to be expressed at a relatively normal level in most cells of the tissue, so any ideas how many ul of starting tissue I'd need?

Many thanks in advance

Robin Harris

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Read the first of only two results

http://search.vadlo.com/b/q?sn=158621799&a...ry+IP&rel=0

HTH

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I just read the title of the topic and believe we could look at the problem in general.

In my lab we have lots of agarose beads, so, I wouldn't buy an expensive anti-HA matrix. I wonder how many mg of protein extract (and at what concentration) I should start from, given that I haven't got a clue on the expression of my protein? As far as I know you're supposed to have 500 uL of, at least, a 1mg/mL extract, but I might be wrong.

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Generally, I use the following considerations.

1. For westerns of unknown quantitiy protein, I start with 50mg lysate/well.

2. For IP of unknown quantitiy protein, I start with 500mg protein/IP.

3. For known High and Low expressing proteins IP, I use 10X of total protein lysate which gives me a comfortable band on western. Which means:

High expression - 10mg lysate on western gives good band?-> 100 mg lysate for IP
Low expression - 100mg lysate on western gives good band? -> 1g lysate for IP

Now, this is my own decision matrix, and have served me well, but you can modify it according to your situation and should still work well.

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