Starting to express protein - tips needed to start it =) (Jan/09/2007 )
Hello there. Need some help again from you guys.
Currently, I have two different constructs. One with pgeX (Amersham Science) and the other one with pqe (Qiagen). Both using different fusion protein. GST for pgeX and His-Tag for pqe.
I know I have to take into consideration of the size of GST and HisTag. Just want to double confirm.
Is GST’s size around 22-29kDa?
Is HisTag’s size around 17kDa?
So if I run SDS PAGE after induction, I have to include those fusion protein into my desired protein right? So the total size will be my protein plus fusion protein.
As for induction part, I tried to follow Sambrook & Russel. There is one part I don’t really get it. I have to load 40ug of each suspension into SDS PAGE. How do I know whether my sample has 40ug? Meaning I have to quantify it before running a SDS PAGE?
Thanks once again. I am new in protein expression.
hello, i think those are correct sizes, yes sure you have to include their sizes in the result of PAGE: protein+GST.
no you dont have to measure the protein concentration. just take some eliquot, i usually take 10ul-20ul to check on PAGE of a mini expression of 2 ml i guess. can't remember the exact now, sorry.
for the previous question: sometimes i do measure my protein before loading using Bradford assay and sometimes i dont , for me it depend on what kind of exp. i am doing.
Thanks Kathy and spanishflower.
Advices are taken. I am using the MiniProtean System from BioRad. But havent really touch SDS before.
Is there another way of quantifying protein instead of Bradford? I read about Lowry too. Which one is better?
the bradford and lowry are both good for the same approximate range of protein concentration. the bradford may be a little more sensitive, the lowry may be a little more linear. the bradford may be more affected by amino acid composition than the lowry is.
you have to decide for yourself which method is better for your purposes.
i do rough estimations. i run some of my protein together with some standard and estimate how much do I have. but of course that is ok for my experiments. it depends.
ah and forgot to tell. i had much troubles trying to dissolve e-coli in SDS-sample buffer received from my freind. Make your own new one and use it. good luck!
Thanks for your kind replies.
I started to express my protein. I am following Sambrook's method. But I don't see any lysing step except for adding SDS loading buffer before loading in the SDS gel.
As from Qiagen handbook, there is some lysing part.
So I am pretty confused. Which one should I follow?
i am sorry cant remember the exact method now, but i think in the sambrook book first you try differnt mini cultures to see which conditions are best: IPTG, timing, temp etc.....for this purpose it is enough to just boil some of the culture in SDS sample buffer. maybe some other protocol tells you to lyse the cells so that protein will be released (go to the soluble fraction), some poeple use sonication in PBS, others use lysozyme.....
however according to my experience it is not so helpful to determine which conditions are best. sometimes your best conditions turn out to be your worst enemy. that is best yeild of protein can get you to inclusion bodies. so my advise just do some minicultures to detemine if your protein is expressed (SDS sample buffer) and then go on to see which conditions give you soluble non-degraded protein that you want. sometimes it is not that complicated though.
Thanks Kathy. I think you are the Queen of the protein. So far I only did twice for expression (one without the lysing part and the other one with lysing as well as protein purification with the help of Qiagen protocol). Haha.. it is pretty much quite tedious.
I guess without the lysing part, it should be enough to determine the protein if expression works since the time expression will show the significant results (i hope). And I guess the SDS component together with boiling will be sufficient to break the cells and release proteins.
I know about sonication used for vigorous lysing. Is there any other chemical methods to lyse the cells? Lysozyme? or to use protease inhibitor? Expression is so complicated! Thank you.
i think you should use protease inhibitor with every cell lysis. all kinds of proteases will be released and they will eat your protein. use a tablet from Roche, it's so convenient. about the method, it depends on your protein. if you get good amount of it in the soluble fraction by sonication in PBS let's say you are the luckiest guy on earth! sonication is cheap, quick and always available. if that fails you try lyzosyme. it really depends what you want your protein for. in many cases you ultimate goal is: to get soluble and non-degraded protein.
so try the easiest and if that doesnt work trouble shoot the problem with more complicated methods like: sarkosyl and Urea (denatures your protein )
forgot to mention: you have to optimize sonication also. number, time of pulses, strenght etc.....ALWAYS put ice in the sonication bath!!!! it should be 4C there.
And I am not the queen of expression. just have worst protein ever that degrades and forms inclusion bodies everytime you decide to touch it!