Serial Dilution and Western Question - (Oct/01/2007 )

Hi all,

I have a quick question regarding serial dilutions and western blotting. I want to do a serial dilution of a purified protein and run it for a western. I have 20ug of protien and I want my dilution series to start at 5ug and continue in a 1:5 ratio until 0.0008ug. I also want to load 10ul per well in my gel. SO, am I correct to do the following:

step 1--Dilute the stock protein @ 1:4 to get 5ug. For example 5ul of stock to 15ul diluent for 20ul total volume.

step 2--Take 4ul of my 5ug and add it to 16ul of diluent to get 20ul of a 1ug solution.

step 3--carry out the dilutions as in step 2 with 4ul sample:16ul diluent until I reach my final dilution of 0.0008ug.

Step 4--Take 10ul of the prepared serial dilutions and use it to load the gel.

If the above method is not correct please advise as to the correct way to do it. Please help!!

Thanks.

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you didnot mention the conc of protein in the stock. I assume that 20ug/20ul.
In that case, your 1st step: take 4ul (4ug) in 20 ul. then you just run 10 ul for WB. Thus, you run just 2 ug.

Other steps are correct!

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Wait....I'm confused! Then what do I do? All I know is that my stock protien is 20ug of protein in a volume of about 20ul. Therefore the concentration is 1ug/ul...right?
Assuming the 1ug/ul is correct then I would do the following:

Step 1: Take 5ul stock protein into 5ul diluent to get 10ul of a 5ug solution.
Step 2: Take 2ul of the 5ug into 8ul of diluent to get 10ul of a 1ug solution.
Step 3: Continue the dilutions in a 2ul:8ul ration until I reach the end of my dilution series
Step 4: Load the remaining 8ul of solution in my gel??

I guess I don't understand how to correctly do the serial dilution and at the same time load the appropriate amount of protein into my gel.

Please help!! I need to do this today.

Thanks!!

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strictly speaking, if you take 2ul of your 5ug, you wont have 5ug anymore.

if what you want to load is 5ug in total, you need your protein at a concentration such as that 10ul=5ug, i.e. 0.5ug/ul, so from your stock (assuming 20ug in 20ul, ie 1ug/ul) take 10ul and add 10ul of diluent (you are doing a 1/2 dilution). now you can dilute this one to make the rest of your samples, i will recomend preparing 15-20ul of each dilution if you want to load 10.
so for example:

step 1: take 10ul protein (stock) + 10ul diluent (this is 0.5ug/ul=5ug if you load 10ul).
step 2: take 4ul of the 0.5ug/ul + 16ul diluent (so this is 0.1ug/ul=1ug when loading 10ul).
step 3: take 4ul of the 0.1ug/ul in 16ul diluent, this will give you 0.02ug/ul=0.2ug if loading 10ul
step 4: take 4ul of the 0.01ug/ul in 16ul diluent, this is 0.004ug/ul=0.01ug if loading 10ul
step 5: take 4ul of the 0.001ug/ul in 16ul diluent, this is 0.0008ug/ul=0.008ug if loading 10ul.

hope this makes sense and helps. good luck!

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V1xC1=V2xC2 where V1 is initial volume and C1 is initial concentration, and V2 is final volume and C2 is final concentration may be useful for you.

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