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Protein expression in e.coli


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5 replies to this topic

#1 neuron

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Posted 23 January 2013 - 01:00 AM

For checking the protein expression in e.coli, till now in our lab we've been doing sonication (for miniscale 5ml culture) , separating out the supernatant and pellet and loading them onto gel. That tells if the protein is present in pellet or in supernatant. But I have seen people using some quick method where you just add some 1-2% SDS or gel loading dye in culture pellet (before sonication), heat it and load on the gel. I am not aware of this. please guide me. How SDS is just enough to lyse the cells, then why do we do sonication?

Edited by neuron, 23 January 2013 - 01:01 AM.


#2 jerryshelly1

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Posted 23 January 2013 - 05:34 PM

SDS/protein loading dye impact negative charges and linearize proteins. I prefer to sonicate and add protein loading dye after (SDS can become frothy and escape tube). Either way will work though.

#3 neuron

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Posted 23 January 2013 - 08:15 PM

Either way will work though.

My question is, sonication is done for lysis of cells. E.coli is having cell wall, so only addition of SDS is enough to lyse the cell? How can we see protein by just adding SDS and heating it? How does it work?

#4 jerryshelly1

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Posted 24 January 2013 - 06:01 AM

Sorry, I misunderstood the question. I think you may be referring to colony cracking. I have never seen it done with SDS by itself, but I have seen NaOH added to lyse the cell. You should look up alkaline lysis and colony cracking. Links are below.

http://kuchem.kyoto-...s/cracking.html

http://labnodes.vand...rce/view/id/711

I hope this helps

#5 almost a doctor

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Posted 24 January 2013 - 08:04 AM

After a quick Google search for "SDS bacterial lysis, wikipedia has some answers Posted Image
By using SDS loading buffer and heat you get total protein but you don't know whether your protein is soluble or not (crude total lysate is very dirty for downstream purification processes, and specially gloppy due to DNA). Is a good quick method to test whether your protein is being expressed at all. When I was doing loads of protein expression, a few years ago, I always used this method. I used to take samples at different time points (from T=0 no expression), then just mix with loading buffer, heat, spin down and load.

and here's the Wiki link, I've copied the part on detergents.

http://en.wikipedia....tergent_methods

Detergent methods
Detergent-based cell lysis is an alternative to physical disruption of cell membranes, although it is sometimes used in conjunction with homogenization and mechanical grinding. Detergents disrupt the lipidbarrier surrounding cells by disrupting lipid:lipid, lipid:protein and protein:protein interactions. The ideal detergent for cell lysis depends on cell type and source and on the downstream applications following cell lysis. Animal cells, bacteria and yeast all have differing requirements for optimal lysis due to the presence or absence of a cell wall. Because of the dense and complex nature of animal tissues, they require both detergent and mechanical lysis to effectively lyse cells.
In general, nonionic and zwitterionic detergents are milder, resulting in less protein denaturation upon cell lysis, than ionic detergents and are used to disrupt cells when it is critical to maintain protein function or interactions. CHAPS, a zwitterionic detergent, and the Triton X series of nonionic detergents are commonly used for these purposes. In contrast, ionic detergents are strong solubilizing agents and tend to denature proteins, thereby destroying protein activity and function. SDS, an ionic detergent that binds to and denatures proteins, is used extensively for studies assessing protein levels by gel electrophoresisand western blotting.
In addition to the choice of detergent, other important considerations for optimal cell lysis include the buffer, pH, ionic strength and temperature.

#6 neuron

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Posted 24 January 2013 - 09:34 PM

@ Jerryshelly1,Thanks for the links. But again you misunderstoodPosted Image , it was not about miniprep. It was for protein. next post by almost a doctor explains what i was talking about. But thanks for the effortPosted Image

@Almost a doctor, Thanks, You got it right, this is what I wanted to know. This method is quick, why dint I know thisPosted Image . I will try this now, just add SDS dye in the pellet, heat, spin and load, right? Is there specific % of sds that we need to maintain?




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