protein expression SOS

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leonando
leonando's picture
protein expression SOS

i have cloned my gene into pGS21a, put it into invitrogen TOP10, did colony pcr and got d correct sized band..so my insert is there right??

 i extracted the plasmid and transformed it into BL21 (DE3) pLysS, BL21 c41 and BL21 c43.. did colony pcr and couldnt get d band...maybe ive put too much colony during d pcr.. so i extracted d plasmid to check if its in there, run it n its there.. i used d purified plasmid to do pcr again but no band except for d plasmid's band... the size of the plasmid increased though...it means my insert is there right?

y cant i amplify my insert then? helpppp

protoldo
protoldo's picture
Hola Leonardo, I never check

Hola Leonardo, I never check the presence of plasmid after transformation in BL21 DE3 pLys. if you have transformed with the correct plasmid, I in your case would do an expressión experiment of any of the colonies in a short volume +/- 5ml runing a PAGE with and withouth induction, seeing the cloned protein. Good luck

leonando
leonando's picture
thanks...ive done it... but

thanks...ive done it... but im not sure if d results r enuf to prove the expression..could u pls check it n give some opinion..

1st pic, 220720104486...
lane 1 -5 = BL21 (DE3) pLysS , lane 6-10 = BL21 (DE3) C41, lane 11-15 = BL21 (DE3) C43

non-transformant, uninduced transformant, 1hr, 2hr, overnight (same for each 5-lanes)

2nd pic, 220720104483 (sample= BL21 DE3 pLysS)
lane 1-4 BSA,  L5=non-transformant, L6 uninduced, L7 20mins, L8 40mins, L9 1hr, L10 2hrs, L11 3hrs, L12 4hrs, L13 5hrs, L14 overnight...

Chin Fen Teo
Chin Fen Teo's picture
 Hi Leonando,

 Hi Leonando,

(1) Do you have a plasmid map (with insert) that allows you to deduce electrophoresis profile for restriction enzyme sites? If yes, I suggest you to perform a restriction digest and see whether the bands are found at where they belong.

(2) What is the Mw of the protein that you tried to express and what conditions have you tried?

leonando
leonando's picture
1. 2 restriction sites in it.

1. 2 restriction sites in it... nco1, ecor5... i did d restriction digest, saw only 1 band.. could it b methylation? coz when i induced it, d prot expressed.. unless there is something else expressed which i doubt it coz i induced (wasted more iptg  :P) d host (non-transformant), d page results r d same as uninduced transformant.. so again, only 1 RE works, methylation on d other RE site??

2. ~40kDa.. 37'c...

protoldo
protoldo's picture
Hola, after summer hollidays:

Hola, after summer hollidays: I have seeing the images and i clearly see an induced band. First ,by the low mobility of BSA your gels are of 12% or more, so I believe  that the band of  wells 6, 10, 11, and 15 of image 4486 is your induced protein. In image 4483 there is a similar  band in all wells from 6 which isn´t in the empty plasmid and appears from non induced untill ON. sample, because depending of strain (Mainly  if it is Lac Iq or not ) and culture conditions you can have the expression partially repressed and an scape of expression could occurs.  Moreover if your protein is a GST fusion, ¿Why you don´t  do a WB, to see which is your protein?. Good Luck