I used to try Ab1 dilution 1:1000 TNT-5% skim milk because it was my first time to detect PIGR, I got nothing but empty film even I used to applied an advance ECL. 2nd, I used to make dilution Ab1 1:500 TNT-5% BSA, and I got nothing too.. till I tried dilution 1: 100 and still nothing there film. I got fast green detection and there is a band I expected as PIGR.. as well I do succeed in DMBT1 detection using 1:100 Ab with TNT-5% BSA. but it doesnt work on PIGR detection. Any suggestions please?
Hello Wenny Ambarsari, it seems like you've been experiencing difficulties with detecting PIGR using different dilutions of your antibody (Ab1) with different blocking agents. Here are a few suggestions to help troubleshoot the issue:
- Check antibody quality: Ensure that the Ab1 you are using for PIGR detection is of high quality and specifically targets PIGR. You can consider verifying its specificity through other detection methods or by consulting with the antibody manufacturer.
- Optimize antibody dilution: Since you've tried various dilutions of Ab1 without success, it might be worth optimizing the antibody concentration further. You can try additional dilutions in the range of 1:50 to 1:500 to find the optimal concentration for PIGR detection.
- Modify blocking agent: If you obtained successful results for DMBT1 detection using 1:100 Ab1 with TNT-5% BSA, it suggests that the blocking agent might not be the issue. However, you can still experiment with other blocking agents, such as non-fat dry milk, to see if it improves PIGR detection.
- Consider secondary antibody issues: If the primary antibody (Ab1) is not the cause of the problem, it's worth investigating the secondary antibody used for detection. Ensure that the secondary antibody is compatible with the primary antibody and is suitable for your experimental setup.
- Assess protein extraction and sample quality: Check the efficiency of protein extraction and sample quality for PIGR detection. Ensure that the protein samples are properly prepared and that the PIGR protein is expressed in the samples you are testing.
- Try alternative detection methods: If the issues persist, you might consider alternative detection methods for PIGR. Immunofluorescence or immunohistochemistry could provide valuable insights and may offer better detection sensitivity.
John Harvey Santos John Harvey Santos John Harvey Santos Thank you so much. I agree with those ideas. I was thinking something wrong with the Ab1, I used to test these protein because it's been there refrigerator 4 degree celsius for 3 years and no one in Lab working on it, another part is I'm thinking that would be no problem since I've done successfully the other protein detection which the Ab1 been kept for 3-4 years too. I'm using the Santa Cruz product [ SC (SPM217): sc-56595 ]. I've done test on IFA too, even the signal was really weak unconditionally.
I am assuming you are using an anti-pIgR antibody. Is it rated to bind the pIgR in a western format? The epitope it detects may not exist in the western blot due to denaturing. Try running the western non-denatured and see if you detect something.
James Drummond Thank you so much, this is also make sense Sir, I've never think this way since previous student was succeed to detect this protein. 1 : 50 also doesn't work, it's nothing coming on my targeted cells. It's been 3 years more in 4 degree C, I would like to test the Ab quality first with the control cell. Best wishes.
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