How to calculate amount of protein to load into Western Blot gel

[wendychang]

Hi All!

I'm having trouble calculating how much volume of my protein samples to load into each well of the gel.

I want to get 20ug of protein per gel, and my protein concentration is around 145 ug/mL. I want to load a max of 20uL of volume( including sds, tcep, etc) into each well.

If anyone can help me, I would greatly greatly appreciate it!

[admin]

Hi Wendy,

how did you analyze your protein concentrations BCA or UV spec?

The calculations are mainly done with dilution calculations.

To load 20 micrograms of protein per gel you should calculate the amount of protein per microlitre you have.

Depending on the sensitivity of your western blot antibodies, and the purity of your protein (also its expression percentage) you may need to load less or more.

You simply calculate how many microlitres you need to reach 20 micrograms, then the rest you can using loading buffer to keep the amounts equal for loading.

Your protein concentration seems low are you sure it is 145 micrograms/mL? That means you have 145ng/microL

Even if you load 20uL of your sample you will only get 145ng/microL X 20 microL = 2900 ng or 2.9 microG you need 10X increase in concentration.

To get 20 micrograms, you will need to concentrate your samples at least 10 X more.

[gehrs]

if your lysate concentration is that low, maybe it would be useful to do a titration of your antibody, just to see the limit of detection. (sometimes is in the data sheet)

and then you could precipitate your proteins to concentrate them all in once.

hope that was useful...

[nawi]

So you have 145 ug/ml (0.145 ug/ul) in your sample..
I am determining concentration od the protein via Bradfrod method..
when you calculate concentration of your sample (0.145 ug/ul), then you have to multiply it with your dilution factor (for example is you have 5 ul of sample in 195 ul of buffer, then your d.f. is 40), and you get the concentration of your sample in 5 ul. If you wanna know how much protein you have in 1 ul of your sample then divide it with 5.
0.145 x 40= 5.8 ug/5 ul , 1.15 ug/ul

If you need 20 ug of protein then divide 20/1.15 ug/ul = 17.39 ul
So you have to put 17.39 ul of your sample on your gel to get 20 ug of proteins..

[proteaHeather]

If you have the equipment available, another option to concentrate your sample would be to dry it down by lyophilizer or speed vac.
If you take a known volume and dry it down, you will know how much protein should be in the dried down material. For example, if you take 500uL of your 145ug/mL sample and dry that down, your dried material would contain 72.5 ug total protein. You could then reconstitute that dried material in ~36 uL solution (water). This would yield a solution that is ~2 ug/uL. You could then take 10uL of this sample and 10uL of loading buffer to give you the total of 20ug protein load.
Hope this helps.

[TorontoStudent]

Yeah, I set up an excel spreadsheet where I enter the concentration of my sample and it calculates how much water and loading buffer i have to add to each amount.

To get the amount you need to do the following calculation.

20 ug/Conc of protein (ug/uL) = uL of sample required.

I am not sure how to concentration your protein since your concentration is so low. But people have given you some pointers with that.