4 replies to this topic

[rpjkmust916]

Hi Forumers

Here I come again, with another problem....please forgive me  

I have this problem in expressing the protein in soluble form. I've played with the IPTG concentration, the temperature during induction and also I've tried using lower temperature which is 22^oC when I start incubate it until reach OD₆₀₀=0.6. Is there any way to solve this? Please..any suggestions and recommendations are appreciated

Attached Thumbnails

• 

[protolder]

rpjkmust916, on 06 April 2011 - 09:56 PM, said:

Hi Forumers

Here I come again, with another problem....please forgive me  

I have this problem in expressing the protein in soluble form. I've played with the IPTG concentration, the temperature during induction and also I've tried using lower temperature which is 22^oC when I start incubate it until reach OD₆₀₀=0.6. Is there any way to solve this? Please..any suggestions and recommendations are appreciated

Hola, I suposse that these images are at +-28ºC  in LB  with 0.2mM IPTG and 3 hours of induction. I think that your protein is tagged ,isn´t it?.What strain do you use? Have any expression before induce? your protein is about 30-40KD. I would use any Origami or Rosetta gami strain for expression, and I would chech any of the internet tools to predice solubility introducing aminoacid sequence if the probability of insoluble form ia quite high, bad thing my friend. Buena suerte

[rpjkmust916]

protolder, on 06 April 2011 - 11:07 PM, said:

rpjkmust916, on 06 April 2011 - 09:56 PM, said:

Hi Forumers

Here I come again, with another problem....please forgive me  

I have this problem in expressing the protein in soluble form. I've played with the IPTG concentration, the temperature during induction and also I've tried using lower temperature which is 22^oC when I start incubate it until reach OD₆₀₀=0.6. Is there any way to solve this? Please..any suggestions and recommendations are appreciated

Hola, I suposse that these images are at +-28ºC  in LB  with 0.2mM IPTG and 3 hours of induction. I think that your protein is tagged ,isn´t it?.What strain do you use? Have any expression before induce? your protein is about 30-40KD. I would use any Origami or Rosetta gami strain for expression, and I would chech any of the internet tools to predice solubility introducing aminoacid sequence if the probability of insoluble form ia quite high, bad thing my friend. Buena suerte

Gracias Protolder

You're almost right!!! I wrote I've incubated it at 22^oC, forgive me to give wrong information. Actually, I've incubate it at 27^oC, with 0.1mM IPTG for 4 hrs. It is a tagged protein with Rosetta strain. Could you explain about the internet tools to predict the solubility. How I can use it?

Muchas gracias

Edited by rpjkmust916, 07 April 2011 - 12:01 AM.

[protolder]

Hola, Now i have changed protein expression and purification for animal pharmacology. These are the tools that I used:
http://mips.helmholt...oso/proso.seam.
biotech.ou.edu, from Oklahoma unv.
refold.med.monash.edu.au
!que tengas un buen día ! (the first is   .de/proso/proso.seam.)

Edited by protolder, 07 April 2011 - 03:06 AM.

[rpjkmust916]

Muchas Gracias Protolder