I need to blot for interleukins which are all less than 50KD what membrane would should be used to blot it appropriately?
Thanking you
We have used 0.2 um Nitrocellulose successfully for cytokine blots (IL-6, IL-10 and IFNg). We run 15 % SDS-PAGE and then transfer using Towbin buffer containing 20 % methanol.
IL-6, IL-10 and IFNg in cell cultures are normally very low (pg/ml range) and cannot be detected with blots. I normally check these using the eBioscience ELISA kits for cytokines. If you use cell cultures secreting high levels of cytokines (higher than 10 ng/ml range) or for recombinant cytokines we run our SDS PAGE first and include rainbow molecular weight markers on the sides. We use a semi-dry blotting system. Normally transfer for 1 hour at constant voltage of 10 V for small gels. Due to low levels of cytokines they cannot be visualized at this stage. Membrane is first blocked with PBS containing 2 % albumin for 1 hours. After that the membrane is used for immunometric detection of the specific cytokines. For detection I use the detecting reagents of the eBioscience ELISA kit. Use at the same concentrations as suggested in kit. The detecting antibody is normally incubated with the membrane at 4 C overnight. Avidin peroxidase incubation is done at room temperature for 30 minutes. Washes are done using PBS containing 0.05 % Tween 20. Four 5 minute washes between steps. I do chromogenic detection of cytokines using TMB insoluble substrate (Pierce-Thermo Scientific).
Dear Sir
It will really going to help me a lot. If you dont mind i would like to ask you many things
initially i did not think on concentration part of interleukin's and thought to do blot, as for now as you said i feel hesitating to do blot however i must learn it
however i like to ask can i develop in house Elisa with available reagents (Primary and secondary antibodies -HRP from cell signalling) Pirce HCL substrate
can i develop in house Elisa with this ?
Have not used the above antibodies yet. In the 90's I developed an in-house ELISA with a polyclonal antibody as capture antibody and a 3E4 monoclonal antibody as detecting antibody. Both were from SIGMA. For signal just anti-mouse conjugated to HRP and then TMB soluble substrate from Pierce (NB:You get both Western Blot and ELISA substrates with TMB). For blocking use 2 % albumin. For detection antibody and conjugate dilution use 0.1 % albumin in PBS/Tween (same as wash solution). For washes use PBS containing 0.05 % Tween 20.
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