I do the western blot to check the anti-GFP antibody.
I transfected the cells with GFP-N1, TIAM1-GFP and VAV2-GFP in HEK293T cell line. When I check the fluorescence microscope, I can detect GFP signal (very strong). However, when I do WB, I only detect GFP-N1 not TIAM1-GFP and VAV2-GFP. Why is that? TIAM1-GFP (235 kD) and VAV2-GFP (130kD).
Note: For TIAM1-GFP and VAV2-GFP: the gene is big so I used the gateway system to clone it. The postdoc in my lab told me that, it could be the GFP of GFP-N1 and TIAM1-GFP are different so that the anti-GFP Ab could only detect GFP-N1. He suggested me to sequence the GFP of GFP-N1 and TIAM1-GFP. What do you think?
Do you have any other comments or suggestions for this?
Thanks
Maybe you have problems with transfer efficiency of the larger proteins. You can try to stain the gels with commassie blue after transfer to see whether you succeeded with the large protein transfer.
If I do ponceau staining after transfer the membrane and do not observe the band, what should I do? I transfer using the kit from invitrogen (iBlot)- dry transfer membrane. It takes only 7 min to finish the transfer step.
There are many of trouble shout in western blot technique so if you see your leader that mean your transfer phase is working. However, you need think about the percentage of your gel so if your protein is small you need increase the percentage of your gel might be work. Sometimes it’s the antibody, for example if you put membrane in secondary antibody for more than 3 hours might be help. If I were you I would increase amount of loading protein more than 60 µg/µl and I would put the membrane in primary antibody overnight and for secondary AB for more than 3 hours. And I would see the loading control if it comes up and you proteins of interesting does not I would use super signal ECL for more than 5 minutes. Good luck
Hello Massar:
1. I can see the ladder in the membrane-very strong
2. I used 100 ug pf protein to run gel.
3. I used 8% of SDS- polyacrylamide gel to run the gel.
4. I incubated 1st Ab overnight and 2nd for 2hrs
5. I used the Licor machine to detect my protein- it will have color (green or red) in the western image. I can observe a huge band of GFP-N1 but not TIAM1-GFP (235 kD) and VAV2-GFP (130kD).
Jiri and Massar:
The postdoc in my lab told me that, it could be the GFP of GFP-N1 and TIAM1-GFP are different so that the anti-GFP Ab could only detect GFP-N1. He suggested me to sequence the GFP of GFP-N1 and TIAM1-GFP. What do you think?
dear Trang
its good idea. However, I would ask the provider first to see what best secondary anti-body suitable for your proteins. and I would think about using super signal ECL to visualize your bands.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line