I need help verifying my calculations prior to proceeding. I have muscle homogenate (a single sample to use as an example) at a concentration of 6.82mg/mL which calculates to 6.82ug/uL.
Now, the antibodies I am using recommend a range of 20-30 ug of protein as sufficient for an adequate signal. Is 20ug, too low? Should I split the difference and aim for 25 ug?
So, if I proceed with 20ug, for the purpose of verifying my calculations, I would need 2.93uL of sample per lane (all gels are run in duplicate). I tend to make double what I need in the event I have to re-run my sample and to account of loading error (I am multiplying this value by 4.6). The result is I need 13.48 uL sample. Using the 1:3 ratio of 4X Laemmli to sample, I require 4.5uL of 4X Laemmli.
Now, per the instructions included with the 4X Laemmli solution from Bio-Rad, "to obtain a final 1x concentration of 2.5% 2-mercaptoethanol (BME, 355mM): add 100uL of BME to 900 uL 4X Laemmli"
Hi there,
Actually reductant is not present in your stock of loading buffer because it is very unstable in time. Therefore the addition of 100µL of BME to 900µL of loading buffer is just made to form the real 4x Laemmli buffer.
Hi,
There are a number of ways to make your sample. Your calculations are correct for diluting the 4x buffer to 1x with 2.5% final BME. But this is what I would do:
Dilute the 4x to 2x with water and 5% BME to make your final working sample buffer
(100 ul = 50 ul Laemmli + 5 ul BME + 45 ul water).
You can make any volume of buffer you need to yield the same proportions, depending on how many lanes you have.
Then add 10ul of this 2x buffer to 3 ul of sample + 7 ul water, heat at 85C for 10 min, pulse spin when cool to pull down any condensate, and load all of the sample on your gel. If you have other samples with differing protein concentrations, you just make them all 10 ul with the addition of water.
All the samples will have the same final volume, and you can just add 10 ul of 2X buffer to all of them.
Always add sample buffer with no protein to the blank wells.The sample for the blank wells would just contain 10 ul water + 10 ul 2X Laemmli, no protein.
If you want to make extra, just multiple all the volumes by the same amount (in your case 4.6), load the number of lanes you need, and freeze the rest.
Hi Geoffery, here in order to load 20 ug in 20 uL by using 4 x Laemmli simply you need to add 3 uL of your sample (2.93 ul)+ 12 ul water + 5 ul Laemmli (BME added). As 4 x means you need 3 parts of sample and one part of Laemmli. It is not the way that adding BME in whatsoever amount will make it 1x. So the x value of Laemmli is only calculated with the sample volume.
Regards...
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