Can someone please help me to calculate protein concentration using BCA standard curve, how to dilute samples and how much 4x Laemmli should I add to denature the protein? I have been struggling for a long time to get into the band.
I have added the current calculation to load an equal amount of protein. I appreciate feedback from the community.
Hi Ranjit Kumar Das ,
First of all, i believe the sample protein concentration is ug/mL (file mentions mg/mL).
I load 50ug protein to check for overexpression in western blot. So you fix a loading volume, say 60uL for all your samples, add 15uL of 4X Laemmli buffer to empty tubes, calculate and add 50ug protein to the respective tubes and make up the total volume to 60uL with lysis buffer if required.
Hope it helps,
Subham.
Hello Subham Basak Thank you much for your note. You meant the ratio of protein lysate and 4X Laemmli buffer would be 3:1 is it. I believe you have gone through my current BCA calculation do you think is it fine or do I need to any modification in the BCA calculation part.
Hello Ranjit Kumar Das
I went through your BCA calculation and did some changes. You need to make the 4X Laemmli buffer constant (1:3 order) and change the lysate and lysis buffer volumes according to the total protein concentration. You need to add lysis buffer in the western blot sample to keep the total protein concentration constant. Here is the modified protocol.
Best wishes
Hi Ranjit Kumar Das ,
Yes, the ratio of lysate and Laemmli buffer would be 3:1, and i don't think you need any modification.
You can follow Satyendra Mondal 's calculations, keeping in mind how much volume your gel wells can hold.
Subham.
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