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Thank you for your reply. I have provided answers to all you questions, noted in red, below.
What is the main objective of these studies? The objective of these studies is to identify BCRP/ABCG2 in 4 different cell lines in order tocooberatewith other experiments that I have conducted. I want to know if BRCP/ABCG2 is present and functional in these cells.
Are these gels have been polymerized under denatured (and reduced) or native and non-reduced conditions? The gels are composed of Acrylamide/bis-acrylamide, %C=2.6% (w/w), %T=Total concentration (w/v) ofacrylamide +bisacrylamide 10 % without SDS, and I run the experimentunder denaturing conditions, withSDS in theloadingand electrophoresis buffers. They are AmershamECLprecast gels that I have purchased from GE Healthcare.
Are the markers pre-stained or not? I use aprestained marker and also an unstained marker. Both can be seen on the membrane but are not visible on the x-ray film. Is this something to be concerned about?
How many times have you run the same experiment under the same conditions? I have ran the same experiment under the same conditions twice. The 4-20% experiments with the first vial of the positive control, where I saw a band near thekDa size of interest, and the 10% experiments with the second vial of the positive control, where the band is double that of the expected band.
Are you using exactly the same samples or prepared freshlysate each time?My celllysates are prepared fresh each time but the positive control originates from the same stock vial.
Have you tried loading only 20ug total protein per lane - instead of 40 ug? Yes I have. I have also decreased the positive control loading concentration to 5 µg.
Thank you for all your help. If you require any additional information please let me know. I look forward to hearing from you.
Asked on Aug 09 2012
Thank you for getting back to me promptly.
It is important to note that the actual (apparent) molecular weight values for the prestained markers can be different from the non-stained markers and also in different gel types.
When a protein is covalently bound to a charge-carrying dye molecule, this can affect the protein's overall charge. Altering the protein’s charge will most likely change its mobility within the gel. This explains why the prestained protein markers are given "apparent" molecular weight values, while regular unstained protein markers are given their true molecular weights.
The apparent molecular weights of certain prestained protein markers can be determined using 10-20% Tris-Glycine gels. It has been observed that when run on different gel types (Tris-tricine, Bis-Tris, etc), the apparent molecular weight seems "incorrect". The reason for this disparity is the different formulations of the gel types (buffering agents, ionic strength and pH).
I would suggest selecting one type of gel and MW markers and use them in the experiments consistently. Rather than comparing the results in between different gel types and MW markers (non-stained and prestained).
I hope this helps and if I can assist any further, please do not hesitate to contact me.
Answered on Aug 09 2012