Affect of pI on protein migration on SDS-PAGE gel - (Mar/18/2008 )

Hi everyone! I am wondering if anyone knows how the isoelectric point of a protein could affect migration patterns of a protein. My protein is around 41.6kDa, but my Western blot gives me a band at around 25kDa. The isoelectric point of my protein is 5.0, so do you think it could make my protein travel faster?
The thing is, I don't know if this band really represents my protein, because I got a band at the same size for my negative control, which I am not supposed to get any bands for. But I have performed immunoprecipitation on these proteins, so I don't see where other proteins would come from and give me this band.
Do you guys have any ideas on what is happening???

Thanks!

Hi everyone! I am wondering if anyone knows how the isoelectric point of a protein could affect migration patterns of a protein. My protein is around 41.6kDa, but my Western blot gives me a band at around 25kDa. The isoelectric point of my protein is 5.0, so do you think it could make my protein travel faster?
The thing is, I don't know if this band really represents my protein, because I got a band at the same size for my negative control, which I am not supposed to get any bands for. But I have performed immunoprecipitation on these proteins, so I don't see where other proteins would come from and give me this band.
Do you guys have any ideas on what is happening???

Thanks!

Hi! I used a Hamilton syringe to load my samples onto the SDS-PAGE gel, and I was told that this syringe does not suck up the beads so I just assumed the it has to be the protein and antibody that's being loaded. But I am not entirely sure how true that is.

But you are right, it could be a non-specific protein that's bound to my antibody. Do you have any idea why isn't my protein that is HA tagged bind to the primary antibody??

QUOTE (Fatleung @ Mar 18 2008, 11:44 AM)

Hi everyone! I am wondering if anyone knows how the isoelectric point of a protein could affect migration patterns of a protein. My protein is around 41.6kDa, but my Western blot gives me a band at around 25kDa. The isoelectric point of my protein is 5.0, so do you think it could make my protein travel faster?
The thing is, I don't know if this band really represents my protein, because I got a band at the same size for my negative control, which I am not supposed to get any bands for. But I have performed immunoprecipitation on these proteins, so I don't see where other proteins would come from and give me this band.
Do you guys have any ideas on what is happening???

Thanks!

I'm not sure about making your protein travel faster. I know that sometimes isoelectric points can affect the way that your protein will transfer onto a membrane. If your negative control is showing a band at equivalent levels then it could be non-specific binding. You can also get non-specific proteins binding to your antibody during IP or it could be binding to your beads. Have you done controls with beads alone - no antibody?

[quote name='Fatleung' date='Mar 18 2008, 01:16 PM' post='129670']
Hi! I used a Hamilton syringe to load my samples onto the SDS-PAGE gel, and I was told that this syringe does not suck up the beads so I just assumed the it has to be the protein and antibody that's being loaded. But I am not entirely sure how true that is.

But you are right, it could be a non-specific protein that's bound to my antibody. Do you have any idea why isn't my protein that is HA tagged bind to the primary antibody??


when I asked if you did the negative control, I meant did you do an IP with just protein A or G beads (no antibody) and do all the washes that you would normally do. If the protein that you are seeing binds non-specifically to protein A or G beads, then it will elute during your boiling step and you should see it in the absence of antibody. As for why you are not seeing your protein - perhaps its not being expressed. You can try some RT-PCR to see if you are getting transcripts made, but you might want to double check your clone first to be sure it was made properly, in frame and all. If you are worried that it might be the antibody that is not working properly then you could try to find a positive control, something that also has HA attached to it and see if you can see that on your blot. Also double check your antibody source. Not all antibodies are good for IP. The literature from the company where you bought it should tell you if it has been tested for IP.

Hi everyone! I am wondering if anyone knows how the isoelectric point of a protein could affect migration patterns of a protein. My protein is around 41.6kDa, but my Western blot gives me a band at around 25kDa. The isoelectric point of my protein is 5.0, so do you think it could make my protein travel faster?
The thing is, I don't know if this band really represents my protein, because I got a band at the same size for my negative control, which I am not supposed to get any bands for. But I have performed immunoprecipitation on these proteins, so I don't see where other proteins would come from and give me this band.
Do you guys have any ideas on what is happening???

Thanks!

Hi you guys!

Thanks a lot for your help.
I know my protein is being expressed because I have performed Western blotting on the yeast and got a band with the correct size (empty vector has no band).
I didn't perform the negative control for the IP you suggested, but I did add protein A/G beads to my sample before I added the antibody to get rid of the non-specific proteins first.
I don't know what's going on, anyways I am doing a third try so hopefully that'll work