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bacterial culture volume for recombinant protein to be detected in Western Blot

bacterial culture recombinant protein western blot volume

Best Answer Missle, 01 March 2016 - 07:11 AM

1.5 mL culture resuspended in 50-100ul of PBS.  If the culture had a typical OD600 (say between 2 and 8) then I would think 10ul would be way too much for WB.  I typically run 5-10ul (max) on SDS-PAGE but would reduce this to 1ul or less for WB.  SDS-PAGE is a good guide though.  Run 10ul on an SDS-PAGE and if you can see the protein of interest, then run 5 ul neat, 5ul @ 1:10 dilution, and 5ul @ 1:50 dilution on WB.  WB is more sensitive than SDS-PAGE but sensitivity is strongly dependent on your conditions (amount of protein expressed AND the sensitivity of the antibody used in detection) so there is really no way to get around looking at a few dilutions....  

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#1 Meg P. Anula

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Posted 26 February 2016 - 07:42 PM

Hi all,

 

How much volume of bacterial culture we can use for recombinant protein to be detected in western blotting?

 

I just wanna test the protein expression in cell unpurified lysates.\

 

Thanks.



#2 Missle

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Posted 29 February 2016 - 05:06 AM

It depends on the amount of bacteria in your culture that was used to generate the unpurified lysates, the protein concentration of the lysate, and - most importantly - the expression level of your protein.  I would do a titration starting at a couple of microliters of lysate and then titering down to at least a 1:100 dilution of lysate.



#3 Meg P. Anula

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Posted 01 March 2016 - 05:56 AM

It depends on the amount of bacteria in your culture that was used to generate the unpurified lysates, the protein concentration of the lysate, and - most importantly - the expression level of your protein.  I would do a titration starting at a couple of microliters of lysate and then titering down to at least a 1:100 dilution of lysate.

 

Let's say for e.coli expression system, usually how much volume of bacterial culture for western blotting detection? My supervisor told me to spin down 1.5 ml broth culture to get the pellet before resuspended with 50-100ul PBS and use 10ul of the mixture for SDS page and WB.

 

But I read somewhere some people starts with 10ml  or 25ml broth culture instead. 

 

I am hoping to have an idea the typical range I should to start with.



#4 Missle

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Posted 01 March 2016 - 07:11 AM   Best Answer

1.5 mL culture resuspended in 50-100ul of PBS.  If the culture had a typical OD600 (say between 2 and 8) then I would think 10ul would be way too much for WB.  I typically run 5-10ul (max) on SDS-PAGE but would reduce this to 1ul or less for WB.  SDS-PAGE is a good guide though.  Run 10ul on an SDS-PAGE and if you can see the protein of interest, then run 5 ul neat, 5ul @ 1:10 dilution, and 5ul @ 1:50 dilution on WB.  WB is more sensitive than SDS-PAGE but sensitivity is strongly dependent on your conditions (amount of protein expressed AND the sensitivity of the antibody used in detection) so there is really no way to get around looking at a few dilutions....  







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