I've been using the Cytoskeleton G/F- actin in vivo kit (https://www.cytoskeleton.com/bk037) on my lysates, and am a stand-still with data analysis. I'm torn between whole protein normalization using the stain free gel technology and comparing to my ECL developed membrane, or if it's possible to quantify bands just using ECL.
My initial thought is that I'll have to do whole protein normalization with the stain free technology since there's uneven loading (and can't use BCA on lysates since the lysis buffer uses b-ME), but then am not sure how to quantify in a way that my samples are comparable to each other.
I do have protein standards (10, 20, 50 ng) and can make a standard curve, but am unsure how to combat the loading issue? The kit suggests to vary the volumes loaded if the bands are outside the linear range, which seems odd to me (some of my samples are 4x my highest standard).
Anyone with experience or insight to this process? Suggestions are greatly appreciated!
Hey Sarah.
is there a particular experimental reason why you have to add ß-Mercaptoethanol directly in your lysis buffer and not later in your Laemmli-Solution?
If you just want to disrupt your disulfide bonds, then this does not have to happen during the cell lysis step.
We lyse cells in commonly used RIPA buffer (150 mM NaCl, 50 mM TrisHCl, 1 % SDS, 0.5 % NP40, 0.1 % deoxicolic acid, without ß-Mercaptoethanol), heat the samples 10 mins at 95°C and store until use at -80°C.
After thawing, we make a BCA Protein Determination (with a commercal kit) and then adjust our samples with water to equal protein concentrations. In dependence on the gel pocket size, we finally add 6 µl (12 Pockets) or 9 µl (18 Pockets) Laemmli (+ß-Mercaptoethanol) to the adjusted samples.
So when you just want to disrupt all disulfide linkages with ß-Merc before SDS-PAGE and Blotting it does not matter during which step you do it. So you can just disrupt them after BCA measurement and not encounter your problem of difficult protein normalisation. If you have a experimental setting, where you want to analyze disulfide linkages between proteins, then of course, this does not work.
I hope that helps you. Please feel free to ask any other question.
All the best,
Marc
If you have access to the stain free gel system with BioRad, I highly recommend that you use this and measure total protein transferred. We have the system and I use it for all of my blots now. As much as beta actin, beta tubulin, and gapdh are considered loading controls, they can change after certain conditions/treatments.
When you measure total protein transferred, it is the exact amount of protein on your blot that you probed with your antibody of interest. I think that this is the cleanest way to analyze data. Plus, it saves time because you have your total protein determined before you probe with your antibody of interest.
In terms of quantification, I use ImageJ from NIH. You can quantify the integrated density for the total protein from each lane making sure that you have the same area for each lane. Then take the integrated density for your protein of interest. Put the protein of interest value over the total protein value.
The integrated density numbers are just arbitrary units and do not have a true value. They are used to compare between bands on a blot. Does this make sense?
Hi, I would suggest to made your own F-actin lysis buffer without beta- Merc., quantify and then add beta-Merc. You can combine your homemade lysis buffer wiht the rest of the kit reagent
Methods:
Article TREK-1 Regulates Cytokine Secretion from Cultured Human Alve...
Maybe you can ask help from someone that use this combination for other reason and use the same kit:
https://www.researchgate.net/post/Which_centrifuge_and_tubes_can_I_use_for_200ul_100_000g_37C_1h_Has_anyone_ever_worked_with_the_G-actin_F-actin_separation_kit_of_Cytoskeletoncom
Hey Marc Herb and Valeria De Arcangelis
Thank you for your responses - I had already lysed my samples and centrifuged and I'm trying to prevent fully re-doing the experiment since my university does not have a micro ultracentrifuge nearby.
Brandon A Kemp
Thank you for the detailed overview of using the stain-free technology for quantification! Would you recommend using Image J for densitometric analysis or BioRad's imaging software (I have both downloaded). I haven't used Image J yet, but BioRad's software gives a normalization value and normalized raw values. I'm wondering if it would be best to increase the range of my standard curve from 10 ng - 300 ng, normalize my samples to whole protein content, and then compare to the standards to get more "real" data.
Currently, we have one generous biochemistry professor I'm working with to see if I can run more gels using her stain-free reagents. My f-actin signal with ECL tended to wash out the signal of the g-actin, so I'm planning to run these on separate gels in the same chamber.
Thanks for all the insight!
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