I am differentiating Rat fetal Stem cells into Neurons. I extract protein for Western blot and quantify them by BCA kit. The problem I am having is after incubation the color of my sample looks similar to a particular standard but when I read the plate using a plate reader the value I get is lower than the zero standard. Someone please help me with this.
BCA absorption must be read at 560 nm, I think the plate reader is not equipped with proper filter. Use spect plate reader or another one. If your standards have correlation then replace the well that your sample is poured into it. The BCA color would be purple.
The plate reader I am using can read 560nm. I should try using other plate reader from other lab. The color is purple, both my sample and the standards. Thank you for answering.
Hi, I try to differentiate stem cells to osteoclasts. I extracted protein at different days. Before doing western blot I need to do BCA assay to determine concentration of my samples. I dont know how to do that and how much of my samples I should run for gel electrophoresis. I really appreciated it if you help me
Sahar Ebrahimi Samani Hi, First take 200micro liter of the BCA solution mixtures. To that add 25 micro liter of standards of different concentrations. Similarly take 200micro liter of bca reagent mixture and we need to add the same volume of our protein like the standards. As we are using 25 micro liters of the standards its not efficient to use 25micro liters of our sample protein as we could not get a lot of proteins. So what I do is I will add 24micro liters of water and 1 micro liter of my sample protein. ie., I dilute my protein 25 times. Mix properly (Use pipette or shaker or rocker) and incubate for 30 mins at 37 degrees C. After that cool it down to room temp for 5 mins and read it in a plate reader at 562nm wave length. That should give you the od value now our standards are 25 micro liter and our protein is just 1 micro liter, so multiply the final value with 25 so that you will get the exact value for our protein. Hope this will help you.
Hi Karthikeyan Vijayakumar !
Did you figure it out? I have a similar problem, but the opposite.
My samples seem (by eye) to correspond to a specific standard, but when reading it, it correlates to a more concentrated one.
Hope you can help me! Thanks!
Mariana Marques, it depends on lots of things. First make sure your standards are in a good condition. If you are not sure about their conditions prepare freshly. Next thing is the plate you are using to measure, make sure you are using a new plate don't reuse plates, that might sometime interfere with the readings. And also make sure the recipe of your RIPA buffer (I assume you use RIPA buffer). The conc of DMSO more than 10% will also interfere with your results. Hope this helps you.
- Badges
- Science topic
- Similar topics
- Indexes