The lung was homogenized immediately in 500ul PBS without the protease inhibitors after removed from the mice. Then centrifuged to collect the supernatant for ELISA. I did the BCA first. The results shows the total protein of lung in the model group was significantly increase compare to the control group. But I haven't detected increased protein level of the target cytokines which were well known to increase in many previous studies and the results even shows a reduction in the model group. I don't know what was happened. The mouse model was well established. Does I ignore some tips which should be concern?
Thank you.
I've always used a tissue protein extraction reagent combined with a protease inhibitor when homogenizing tissue for tissue lysate ELISA. I don't have experience with lungs but have had success detecting cytokine in skin homegenates. Pierce makes T-PER cat#78510.
Protease inhibitors...... and since you have two whole lungs per mouse, why not take part of one for qRT-PCR?
good luck!
G
ps - more overall protein per unit volume.. were the lungs congested with serum? was their a weight difference?
Hi Dominic, Thank you for your suggestion! As the lung was freshly removed from mice, I think maybe the protein can not be degraded in a short time. So the protease inhibitors were not used. I haven't dilute the lung homogenate. Have you detect the T cells cytokines in Lung? And what a dilution you prefer? Thanks!
Hi Grant, Yes, the other part of lung was used for Q-PCR. It will be better if i get a good protein results. I wash the lung with PBS to get the blood away on the surface of the lung. But I haven't removed the inner blood of the lung. The whole upper lobe of the left lung were homogenized each mouse. I haven't weight them. Maybe there are some little difference.
Jiaoyan - it's always better to use protease inhibitors, just for future reference. For ELISA, we tend to use the eBioscience kits - the quality and reproducibility is very high. As Dominic suggests, 1:10 is a good start, but don't be surprised if some cytokines - IL-6 and IL-8 for example, "top out" your assay and you have to dilute them further, while others, IL-4 and IL-13 for example, may be very weak signals.
Good luck
G
Hi Dominic, Grant, Thank you guys! Good reference and suggestions! I will try it again!
This question has been very helpful for my project. I'm currently investigating the levels of IL6, VEGF and TNFBeta 1 in mice tissues after treatment.
I had to do a series of dilution to test what dilution works best for ELISA kits for IL6 from mice tissues. Could anyone give me a estimate? I read and it's about 1:20 dilution.
What about dilutions for VEGF and TNFBeta to use for ELISA kits for mice skins?
Thanks
I am working on extracting cytokines from mice lungs in order to quantify them by the Biorad multiplex kit. Is there any additional step to extract cytokines from lung or I can extract them using the same protocol of protein extraction?
A lysis buffer and protocol are recommended by Biorad (see the attached file), they recommend to use just the PMSF as a protease inhibitor, is it sufficient? Because I want to use a part of the homogenate to perform western blot later.
Do you recommand using another lysis buffer and add a protease cocktail inhibitor too to my samples?
Last question, since I will be testing different cytokines in every sample at the same time, can anyone give me an estimate about the dilution?
Thankss!
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