Membrane Protein Western Blot.
Posted 01 December 2004 - 12:32 AM
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!
Posted 01 December 2004 - 06:24 AM
Posted 02 December 2004 - 08:34 PM
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...
Posted 03 December 2004 - 12:51 AM
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!
Posted 03 December 2004 - 06:09 AM