Hello Laura,

If you are looking for expression levels of a recombinant protein, you should be able to check/find sequence information and easily compare it to E.coli database. By doing so you can eliminate house-keeping genes. If your protein of interest is expressed, you should be able to pull out only that protein by using your antibodies in WB. (if they are normalized well & highly specific)

hope this will be helpful,

sincerely,

irem

You can add whole cell lysate which dosent have your protein, another well behaved housekeeping protein which will serve as an another control with 2 loading amounts ( Say 10 and 20 ug) , and then various amounts of your protein of interest ( Here include 10 and 20ug) and do the western blot. Compare ... Thats it

Dear Laura

of course yiu need to load a similar amount of cell lisate for each line, however generally if you are looking to the expression of a recombinant proteins for purification, the protein have to be over expressed in goid amount and therefore your normalizzation could be rude. you can just measure the od of the culture, lisate and run in the gel a fix od of lised cells ( i suggest to yuo to run both total and solubile fraction)

generally i load about 0.08-0 12 od (600nm) for lane. (see https://www.researchgate.net/publication/315999127_Multiwell_based_protein_expression_and_solubility_test)

if you want to be more precise, you can quantify your extract with bradford or bca and load a costant amount (e.g 20ug) for lane but this is more important when you are performing wb analisis of cell lisate for check the expression of a constititive gene if the cells.

if the expression is not clearly detectable because your protein run a mw where the non induced e.coli show other strong bands in sds page, if you clone contain a his, gst or mbp tag you can perform or a wb with anti tag antibody or a small scale purification with gravity flow coloumn (using 100ul of resin) https://www.researchgate.net/publication/314950177_SMALL_IMAC_MANUAL_PURIFICATION_a_simple_way_to_purify_manually_up_to_24_his-tagged_protein_samples_in_parallel_up_to_1mg_of_purified_protein)

good luck

Manuele

If you just want to know whether your recombinant protein is expressed, you can run SDS-PAGE for a same amount (ex: 20ug) of whole cell lysate of cells that potentially express your protein (experiment) and parent cells (control; don't express your protein). Then, you should be able to see a thick band around the M.Wt of your protein in the experiment lane compare to the control lane. That suggests your protein has been expressed. If your protein is expressed after induction (like adding IPTG), you can run SDS-PAGE for whole cell lysate of before and after induction. Good luck.

If this is only to evaluate the over-expression of your protein, you can can harvest your cells by centrifugation, than you can lisate your cells by sonication.

For example, for an OVN cell culture of 10-50 ml of saturated culture medium, you could centrifuge the cells, then wash the pellet with whatever buffer you want, then you can resuspend your cellular pellet with whatever buffer you want to use (preferably a pH of 7). When you re-suspend your cells you need to make sure that you use an adequate amount of buffer, not to much so that your cells are to diluted and not to little that you have a concentrated amount of cells (I aprox. use a 20 ml of buffer for a 5 ml medium culture). Once your cells are resuspended, you can sonicate your cells. Do this in an ice bath, and then you can centrifuge your cell lysate to separate the membrane part of it from the cytoplasmic part. You can do a BCA or a Lowry-TCA to both parts of your cellular lysate sample to quantify your protein. Then load 10, 15, 20, etc., ug of protein of each sample (membrane or cytoplasm) (this is to evaluate if your protein is in the soluble part or if it might be going into inclusion bodies, or if you are over-expressing a membrane protein) so that you can evaluate how much protein you need to load and stain with coomassie. In your WB you can use a housekeeping gene as a loading control to corroborate the amount of protein you are charging, for example, you could use the antibody that will recognize the Beta subunit of the ATP synthase, it worked for me in different growth conditions and in different bacterial strains.

As it has been commented before, use a control of cells without induction to evaluate the amount of over-expression you are achieving by comparing with your inducted cells.

Good luck with your experiments!

Figure 5 B

Article The Biological Role of the ζ Subunit as Unidirectional Inhib...

Dear all,

thank you for these precious suggestions!

Sincerely,

Laura

weight your cell pellets and calculate the loading amalogies