Hi I've been trying to produce some recombinant human Interleukin 6. I sub-cloned the gene for human IL6 into a Promega T7 Flexi vector (native gene no tag yet) and confirmed sequencing and restriction enzyme digestion. I have now induced expression in a BL21 E.coli culture with IPTG and also used chaperone proteins to increase soluble IL6 (as suggested in literature). Grown at 37C with shaking until OD 600nM=~0.5, IPTG added and then temperature dropped to 22C and culture is left overnight. Protein is then extracted with bug buster, as its quicker for the small scale and Roche protease inhibitor cocktail added.
It looks like its been successful on the attached image (expected size of IL6 is 21kDA) but I keep getting some non specific bands on my western. I was wondering if anyone knew what they might be?
I'm using the following primary antibody at 1:1000:
https://www.abcam.com/il-6-antibody-epr21711-ab233706.html#description_images_4
Secondary antibody antibody at 1:20000:
https://www.abcam.com/goat-rabbit-igg-hl-alkaline-phosphatase-ab97048.html#description_images_1
Blocked with 3% BSA, antibodies also in BSA and washed with TBST (x3 15mins each).
We don't have a western blot camera in our lab so I've had to use an alkaline phosphatase conjugated secondary antibody and develop with BCIP/NBT.
The only thing I can think of is perhaps there is some cellular protease activity going on which has created fragments of IL6 that are showing up on the western.
Any help you can give would be appreciated as my experience with recombinant protein expression is limited.
*The aim is to produce a large amount of IL6 so we can examine it with neutron beam small angle scattering. It's related to COVID19 symptoms and the resulting cytokine storm IL6 is thought to be involved in.
Hi there,
Degradation might be responsible for the lower bands. As you have already tried the addition of anti protease cocktail in your lysis buffer (complete from Roche), it tends to prove that if it is degradation, it either occurs within the cell or during extraction and it's not inhibited by complete mix...
Yes the inhibitor was added to the lysis buffer. Maybe the degradation occurred since the culture was left overnight, maybe 19 hours total. This possibly could be avoided by using a protease inhibitor that could be added to the live culture?
Another possibility (for the smaller non-specific products) is incomplete translation products that still contain a portion of the antibody binding epitope, as you are doing a whole cell lysate. Does your construct have a signal peptide that directs the protein to the periplasmic space (e.g. PelB, OmpA, DSBA)? If so, you could try a periplasmic extraction, it'll yield less protein, but should in theory yield only completely synthesized protein that should clean up the smaller non-specific products in the Western.
https://www.abcam.com/il-6-antibody-epr21711-ab233706.html#description_images_4 Abcam also say there is non-specific binding to a product at 40 KDa in their QC western.
Hope this helps
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