My current protein concentration is 13mg/ml. To load 30ug protein for SDS PAGE, only 2.15uL is needed, can i load 2.15uL sample + 2.15uL 2X Laemmli Buffer = 4.3uL in the well or do I dilute the sample first so that more volume is needed?
If so, how do I dilute the cell lysate, or can I just use PBS?
Help!
I diluted my proteins in pbs. It works just fine. Alternatively, you can dilute them in your lysis buffer so that they will have final concentration of 3ug/ul. In that scenario, you can load 10 ul for each well and they all contain 30 ug protein.
It can help a lot to dilute cell lysate samples, as they can be very viscous due to their high protein and cell biomass content.
I usually aimed to add 10 uL of sample to a 20 uL SDS-PAGE sample well.
E.g. in your scenario I would prepare a tube containing
4.3 uL sample,
10 uL 2x Lamelli Sample Buffer,
and then make it up to 20 uL by adding 5.7 uL water (you can use 1x PBS, TBS, whatever, instead of water, if you are concerned about diluting out the salts).
Now load 10 uL of this and it will contain your 30 ug protein, but I found it easier to pipette.
It also yields an extra 10 uL in case of error.
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