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[plasmodel]

So, I am trying to express a parasitic protein in E. coli.

I used pET-28b to get N-terminal and C-terminal His tags (ie. clones have either C-terminal or N-terminal not both). I then used BL21(DE3) cells to grow them and induced using IPTG (1 mM or 0.5 mM) at 37C. Anyways, my question is in the past when I have done this (in lab classes) we basically just took the same amount of samples at various time points (eg. 1 hr, 2 hr, 3 hr etc) and then, used that pellet with SDS-sample buffer to run an SDS-PAGE.

My question is: Does one need to make sure that the samples taken out of the culture at the various time points are at the same concentration, so that one can run a gel where all the samples are at the same concentration? I have done this by diluting the samples such that all of them are at the same OD more or less. But, I don't seem to be seeing anything (I just same strength bands)........But, I feel like I should make sure they are the same concentration because later stage culture samples will obviously have more cells---and the protein expression band will be stronger just because there are more cells expressing this protein.

ALSO, I started using this new anti-His tag antibody (IgG, pclonal, rabbit) and I basically see in all my induced samples multiple bands and not just the one I expect....I even see my ladder (with more bands than the normal ten on a protein ladder). I tried different blocking solutions (5% milk; gelatin); various concentrations of the commercial antibody (1:250 (recommended) and 1:5000); various washing steps (4X5'; 4X10')...but, still lots of bands and even, the ladder shows up!!!

HELP!!!!!!!!!!!!!

[protolder]

plasmodel, on 07 January 2011 - 03:20 PM, said:

So, I am trying to express a parasitic protein in E. coli.

I used pET-28b to get N-terminal and C-terminal His tags (ie. clones have either C-terminal or N-terminal not both). I then used BL21(DE3) cells to grow them and induced using IPTG (1 mM or 0.5 mM) at 37C. Anyways, my question is in the past when I have done this (in lab classes) we basically just took the same amount of samples at various time points (eg. 1 hr, 2 hr, 3 hr etc) and then, used that pellet with SDS-sample buffer to run an SDS-PAGE.

My question is: Does one need to make sure that the samples taken out of the culture at the various time points are at the same concentration, so that one can run a gel where all the samples are at the same concentration? I have done this by diluting the samples such that all of them are at the same OD more or less. But, I don't seem to be seeing anything (I just same strength bands)........But, I feel like I should make sure they are the same concentration because later stage culture samples will obviously have more cells---and the protein expression band will be stronger just because there are more cells expressing this protein.

ALSO, I started using this new anti-His tag antibody (IgG, pclonal, rabbit) and I basically see in all my induced samples multiple bands and not just the one I expect....I even see my ladder (with more bands than the normal ten on a protein ladder). I tried different blocking solutions (5% milk; gelatin); various concentrations of the commercial antibody (1:250 (recommended) and 1:5000); various washing steps (4X5'; 4X10')...but, still lots of bands and even, the ladder shows up!!!

HELP!!!!!!!!!!!!!

Hola, adjusting all the samples to the same O.D. is a good way to standarize the amount of protein in each well.About expression, for me the problem is that the strain don´t repress expression and there is a constitutive one without inducer. If you have, try one of BL21 lacIq or with yours add 0.2g/l of glucose, grow ON at 25-28ºC and check expression without induction in order to see the sole band that you want. block at least 2h. at RT and if you continue seeing multiple bands, I don´t know how to help you because the increase of dilution rate is ,for me,the correct way to eliminate inespecific bands. Buena suerte