21NOV17 IRM13 protein concentration calculation

21NOV17

I need to calculate my IRM13 protein concentration so I used the Qubit estimation first, the Absorbance method at 280nm and the BCA method.

Qubit

Qubit estimated that I have 202 ng/μl

Absorbance at 280nm

- switch the spec on
-Program Scan in the PC
-Set up
-Accessories
-Click use cell charger, cell1 cell2
-Click Baseline, baseline correction
-Cary, start 400 stop 200
-Scan control - medium
-OK
-OK
-Click the baseline
-Click zero

I placed my blank (which is the flow through buffer when i was concentrating my protein down) in cell 1 and my sample in cell 2.

-Click Start
-Name the file
-Right click on the graph, peak labels, x and y labels, apply
-If the peak at 280nm is not recognised then right click on the graph, cursor mode and track


My y values is 0.247512. The extinction coefficient of IRM13 (according to Expasy) is 87015. So according to Beer_Lambert law Α = ecl I can calculate the concentration

c = A/el
c = 0.247512/87015x1(pathlength)
c = 2.844 x 10E-6M
c = 2.844 μM

Based on the formula

M (mM) = C (mg/ml)/kDa
C = M x kDa
C = 2.844 x 10E-3 x 90.851
C = 258.38 x 10E-3
C = 0.28538 mg/ml
C = 285.38 ng/μl


BCA method

This is a method which is time sensitive so it needs to be done quickly and be prepared for the next step. I followed the instructions in the SOP preparing the standards using BSA and buffer used for diluting my protein (I used the collected flow through when I was concentrating my protein after dialysis) and serial dilutions of them.

I mixed the required amount of buffer A and B and I multipipette the mixture. I incubated for 30min at 37oC and then measured at 562nm

I used an uploaded excel template for automatically calculated the protein concentration. I followed the instructions.

BCA tells I have 51.8μg/ml
51.8 ng/μl


I decided to trust the BCA results
I stored my protein at -80oC in 20% glycerol