Hello everyone!
I am currently having quite a hard time trying to detect IL-18 in human serum by Western Blot and I honestly do not know what can I improve in my technique or what can I modify so I can obtain better results...
Currently, I am performing the protein separation (total serum protein in each well >200 ug) in a 12% SDS-PAGE gel at 110 V for 1 hour, then I do the transfer for 1 hour in a Biorad transfer cell (wet protocol in transfer buffer containing no SDS and 10% methanol, 100 V, ~300 mAmp) followed by membrane blocking (1 hour) primary antibody exposition (3 hours, 25°C, mild shaking) and secondary antibody exposition (HRP-conjugated, 3 hours, 25°C, mild shaking).
I develop the membranes with ECL reagents and have tried detection in Biorad Chemidoc MP digitalizer using SAM mode and in traditional X-ray film but I cannot detect a single band...
Any idea of what can be going wrong? Any suggestion you could have?
I really appreciate all of your help and assistance!!
Best,
Andrés.
Dear Andres,
Try changing the amount of protein may be a little less and human serum contains other higher molecular weight proteins such as albumin which may hinder your results. You should also know your abundance of the target protein in your sample. According to the molecular weight of the protein you can use the gel for example - 4-12% gradient gel for better separation and uniform migration of the protein or just use uniform 10% gel. you can also do the initial run of the gel at 4 degrees. Check for the primary antibody and secondary antibody specificity and their concentrations or you can perform dotblot to know whether your antibodies work. Over night incubation of primary antibody at 4 degrees. One hour incubation of HRP secondary antibody. Hope you run an internal control so that you are sure about the results that you get. Hope it helps.
Use these links: https://www.abcam.com/protocols/western-blot-troubleshooting-tips.
https://www.rndsystems.com/resources/technical/troubleshooting-guide-western-blot
I would first suggest to check whether the IL-18 is present in the serum or not. Dot blot is a good idea to check the specificity of the antibodies as suggested by Vishnu. For all other troubleshooting I would suggest to run western with another set of antigen and antibodies. I think the problem lies in the presence of IL-18 and if present, check the antibodies and their concentrations to be used for a proper detection.
Hi Andres,
These could be some of the tips which you can follow up..,
1. I would recommend you to perform DC assya to check for exact amount of proteins in your serum sample.
2. Use 1X running buffer and 1X transfer buffer.
3. If you are using PVDF membrane, then better to soak it in alcohol for at least 1min.
4. After the transfer check the bands using ponceau staining which will tell you about the transfer of proteins on to the blot.
(If you could see the bands then you can proceed with antibodies. or else, check these steps once again!!!).
5. Use 1:500 or 1:100 dilution of primary antibody. Better to prepare the antibody in the blocking sulution!!! and and and it is very much needed to keep the blot with primary antibody overnight at 4'c.
6. Make sure that you used proper secondary antibody (if primary antibody is raised in mouse then you should use mouse secondary antibody!!!!)
7. Make sure that there's very less time gap between the ECL treatment and taking the image. Ideally it should not be over 5 minutes.
Hope this would help.
-- Best regards.
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