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Thank you for the second vial of my replacement positive control (ab29743).
I have been using the second vial of the positive control provided in my recent experiments (5 and 10 µg total protein per lane after denaturing for 5 minutes at 95C, as stated in the product data sheet) with BCRP/ABCG2 primary antibody ab63907. I have attached images of my blots for your information.
I have encountered another issue!! As you can see, I have used two types of precast gels: a 4-20% gradient gel and a 10% homogeneous gel both from Amersham, but I get contrasting results which are worrying and confusing me. I detect a band at61.7kDa in 10 µg positive control and 63.1kDa in 20 µg positive control (on slide 1. This is the slide that I had previously shown you in an earlier email) on the 4-20% gel, whilst on the 10% gel I detect a band at 138.0KDa (slide 2). I'm very confused with these results and I was wondering if youwould have a possibleexplanation for this or solution? Do you think that the positive could bedimerised in the second vial?
I continue to usea rabbit polyclonal toGAPDH (ab37168) as a loading control, and as you can see on both gel typesI seeGAPDH in the expected region.
I have also repeated these conditions twice per gel and I'm getting the same results.
I hope you will be able to advise me on what I can do next. I used the same experimental conditions that I stated in an earlier email to yourself.
Thank you in advance for your help.
Asked on Aug 08 2012
Thank you for getting back to me and for sharing your results with me.
The storage buffer of the positive control lysate contains beta mercaptoethanol (reducing agent) so it is very unlikely that the protein is dimerized particularly if the WB was carried out under denaturing conditions.
Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
What is the main objective of these studies?
It may be difficult to compare the results with 4-20% gradient gel vs 10% homogeneous gel head-to-head. Are these gels have been polymerized under denatured (and reduced) or native and non-reduced conditions?
I would recommend selecting/using only one type of gel. BCRP/ABCG2 can be separated on 10% or 12.5% gel so you would not need to use 4-20% gradient gel otherwise it is rather hard to interpret the results.
Protein size (kDa)
Gel percentage (%)
The MW markers can’t be seen on the 10% gel and it seems that the other picture only has the annotated MW numbers but not the actual MW bands. Are the markers pre-stained or not?
How many times have you run the same experiment under the same conditions?
Are you using exactly the same samples or prepared fresh lysate each time?
Have you tried loading only 20 ug total protein per lane - instead of 40 ug?
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.
Answered on Aug 08 2012