Membrane Protein Western Blot. - Troubleshooting western blot (Dec/01/2004 )
Hi there ,
I've heard that membrane proteins cannot be boiled before loading to a SDS-PAGE for western blot. Does anyone know why is that so? I'm dealing with ABCG2 tissue protein and all the while, I had nice bands showing at 72kD. However, when I'm doing a verification for the previous results, I can't detect the band-of-interest. The only thing I had changed is probably increasing the temperature from 95oC for 5mins to 99oC for 10mins. This goes to the same for my positive control (cell line), yet I got a band for it.
I had tried re-extracting proteins from the same samples, thinking if the previous results were due to the degradation of my protein, but I still can't get the band out.
Can anybody please help? Thank you!
hello, i am dealing with ABCG2 too. but i don't do western blot. what kind of antibody did you use for the western blot? is the positive control sample prepared again by yourself or did you just use the previous remainder? the monoclonal antibody against ABCG2 transformation or any peptides epitope, or any polyclonal antibody against somewhat peptides epitope? i suggest you should prepare enough membrane proteins (including your positive control sample) for at least twice western blot detection and see if you can repeat your desired result at first. if you do, then move on to another sample protein preparation for verification. it's normal not to repeat our result.
I used the previous remainder for the positive control. I'm using a monoclonal Ab for that and I actually had used it for a number of times to do western blot and it works all the time.
The problem is that I don't understand why I can't get back the 70kD size since I'm using the same old sample. I'd tried extracting new tissue samples to see if my samples had degraded, and it proves that my samples have not. I'm wondering whether if the temp for boiling'll affect the aggregation of the membrane proteins as diccussed in some other forum. Hmm...
I finally got back my band-of-interest! That's when I didn't add any reducing agent to the SDS sample loading buffer. Does anyone know why is that so? If there's no reducing agent added to the proteins before boil, can I confirm the band that I see is the protein that I'm hybridizing? Thanks!
the epitope recognized by your anti-ABCG2 monoclonal antibody is probably the conformation but not some short peptide sequence. so it makes difference that when you added the reducing regent to your protein extract which will disrupt the S=S and chang the conformation of ABCG2 transporter, eventually result in the failure of your western blot. by the way, the anti-ABCG2 monoclonal antibody doesn't work on fixed cells in immunofluorescence.