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cytoplasmic and nuclear extraction for BCA


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#1 Nicky

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Posted 25 November 2010 - 09:20 AM

Hi group

I'm new to this forum and would appreciate for your kind help. I'm using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit from Pierce to extract the nuclear and cytoplasmic protein from soft tissue. Subsequently I'll run a microBCA (Pierce) to measure the amount of proteins from these two extracts.

I have two questions:

(1) To run the microBCA, I need 150 ul of each standards and diluted samples (i.e., 300 ul for each replicate). With the tissue that I work on, I need to dilute the extracts in order to get such volume. What reagents should I use to dilute both nuclear and cytoplasmic extracts? It may be most straightfoward to simply use the extraction reagents (CERI/II for cytoplasmic extract and NER for nuclear extract)as provided in the kit. However, these reagents only come in limited volume and I would like to save them if there's an alternative.

(2) A related question: the microBCA manual suggested to make the standards using the same diluent of the samples. If I use CERI/II and NER as diluent for cytoplasmic and nuclear extracts respectively, do I need to run standards for each diluent? The same concern as above is that if I use these reagents as diluent for making standards, I basically use up one kit for a plate. I wonder if there're any other diluents that I can use for making standards.

Thanks a bunch!
Nicky

#2 bob1

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Posted 25 November 2010 - 04:25 PM

1) if the nuc and cyto extracts are the same volume and the same buffer, then you can dilute in another solution (e.g. PBS, water, TBS) so that the final concentration of the extraction buffer components (not the protein though) is the same. If they are in different buffers, you should run a separate set of standards using the different buffers for blanking.

2)You should use the same buffer as in the kit if you can otherwise there may be something in the extraction buffer that interferes with the BCA giving you a false protien concentration.

If you did measure using a different buffer for the standards, assuming that both the extraction buffer and the standard buffer have linear curves with the same slope (you could test it once to see if this is the case), you could measure relative to the standard buffer. It won't give you an exact measure of the concentration, but it should give you values that are comparable across all samples.

If you need more explanation I'll draw a graph and hopefully get the idea across.

#3 Nicky

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Posted 25 November 2010 - 08:37 PM

Thank you so much, Bob1. The nuc and cyto extracts will be in different buffer. The cyto extract will be in a buffer made up by CERI and CERII, whereas the nuc extract will be in NER. And these two extracts will be in different final volume because according to the manual, the ratio of these three reagents has to be 200:11:100.

But is it still possible to dilute the extracts using PBS etc for the microBCA? Would you mind drawing a graph?

Thanks a lots!
Nicky


1) if the nuc and cyto extracts are the same volume and the same buffer, then you can dilute in another solution (e.g. PBS, water, TBS) so that the final concentration of the extraction buffer components (not the protein though) is the same. If they are in different buffers, you should run a separate set of standards using the different buffers for blanking.

2)You should use the same buffer as in the kit if you can otherwise there may be something in the extraction buffer that interferes with the BCA giving you a false protien concentration.

If you did measure using a different buffer for the standards, assuming that both the extraction buffer and the standard buffer have linear curves with the same slope (you could test it once to see if this is the case), you could measure relative to the standard buffer. It won't give you an exact measure of the concentration, but it should give you values that are comparable across all samples.

If you need more explanation I'll draw a graph and hopefully get the idea across.



#4 bob1

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Posted 26 November 2010 - 03:33 PM

Ok, attached is roughly what I mean. The examples are totally made up, so only for explanation purposes and may not actually resemble what the graphs would look like in real life:

The Blue line is the calibration curve done using the lysis buffer to dilute the standards, so this would give the correct concentrations if you calibrated your samples off it.

The pink line is one example of a calibration curve using another buffer as diluent instead of the lysis buffer - the concentrations of your samples in lysis buffer would be out by a set amount that doesn't vary across the whole calibration range, and would be OK to use as it is consistent.

The yellow line (I hope you aren't blue/yellow colour blind) is what could also happen if you dilute your standards in a buffer that is not the lysis buffer - but it will give you different values for the concentrations of your samples across the calibration range, overestimating the samples that are at the low end, and underestimating those at the high end. As you won't have a calibration curve generated using the lysis buffer, you won't know this error until you run the gels and get funny results.

Hope this makes sense.

#5 Nicky

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Posted 26 November 2010 - 04:38 PM

Thanks heaps, Bob1! Somehow I didn't see the attachment from your last post. Do you know where I can download/ view it?

#6 bob1

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Posted 28 November 2010 - 03:05 PM

Oops, sorry didn't manage to attach it properly - I just put it into the attachments box and forgot to click "Attach this file".

Attached Files



#7 Nicky

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Posted 28 November 2010 - 03:27 PM

Got it, Bob1. totally make sense to me. Thanks a bunch!




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