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Expression problem using invitrogen pRSET C vector


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5 replies to this topic

#1 valarie

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Posted 19 December 2009 - 05:00 AM

Hi all,
I am having trouble expressing my recombinant proteins using pRSET vector and BL21 DE3 host.
Conditions:
25 deg C 0.5mM IPTG, 1.0mM IPTG
30 deg C 0.5 and 1.0 mM IPTG
Running at 12% SDS PAGE and Western blot shows no bands.

Optimization of Western blot using 1: 2500, 1: 2000 primary Ab incubated for 2 hours as well as overnight incubation shows no bands.
Blocking was tried at 5% skim milk overnight, and 10% skim milk at RT 2 hours shows no bands.
When positive control was included, bands can be seen clearly, so its no supposed to be antibody problem.

Double confirmation of sequencing results show that clonning was correct and in frame.

Whats the reason that i still coundnt get my recombinant proteins expressed?

#2 Vini

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Posted 19 December 2009 - 06:25 AM

Hi all,
I am having trouble expressing my recombinant proteins using pRSET vector and BL21 DE3 host.
Conditions:
25 deg C 0.5mM IPTG, 1.0mM IPTG
30 deg C 0.5 and 1.0 mM IPTG
Running at 12% SDS PAGE and Western blot shows no bands.

Optimization of Western blot using 1: 2500, 1: 2000 primary Ab incubated for 2 hours as well as overnight incubation shows no bands.
Blocking was tried at 5% skim milk overnight, and 10% skim milk at RT 2 hours shows no bands.
When positive control was included, bands can be seen clearly, so its no supposed to be antibody problem.

Double confirmation of sequencing results show that clonning was correct and in frame.

Whats the reason that i still coundnt get my recombinant proteins expressed?



Hi

did u chk whether ur protein is going in pellet??? Maybe u should try more conditions....did u try at 18 C? and check with uninduced culture as well

#3 valarie

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Posted 24 December 2009 - 09:57 PM

Hi all,
I am having trouble expressing my recombinant proteins using pRSET vector and BL21 DE3 host.
Conditions:
25 deg C 0.5mM IPTG, 1.0mM IPTG
30 deg C 0.5 and 1.0 mM IPTG
Running at 12% SDS PAGE and Western blot shows no bands.

Optimization of Western blot using 1: 2500, 1: 2000 primary Ab incubated for 2 hours as well as overnight incubation shows no bands.
Blocking was tried at 5% skim milk overnight, and 10% skim milk at RT 2 hours shows no bands.
When positive control was included, bands can be seen clearly, so its no supposed to be antibody problem.

Double confirmation of sequencing results show that clonning was correct and in frame.

Whats the reason that i still coundnt get my recombinant proteins expressed?



Hi

did u chk whether ur protein is going in pellet??? Maybe u should try more conditions....did u try at 18 C? and check with uninduced culture as well


I hv tried at 20 deg C as well...it still did not show any bands on western blot... For SDS PAGE, when compare with the uninduced and untransformed control, the bands were the same (no distint and thick bands that could differentiate between them).
The proteins were to be soluble form, and i used the total cell crude proteins for identification. It still turned out to be nothing....

#4 Vini

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Posted 25 December 2009 - 04:07 AM

how long do u kp the culture after induction???

#5 valarie

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Posted 26 December 2009 - 10:14 AM

how long do u kp the culture after induction???


the next day i used it for SDS PAGE and western blot

#6 Vini

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Posted 27 December 2009 - 01:02 AM

how long do u kp the culture after induction???


the next day i used it for SDS PAGE and western blot



Does that mean 12 hrs.???? If thats so, try increasing the time to 16-18 hrs.....although, if u r using Kanamycin as the selection marker, dont keep for more than ~14 hrs. Also, is your gene AT or GC rich???? in that case, u may want to try the expression in some other comp. cells. Like RIL-strain for AT-rich, RP strain for GC-rich etc......




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