Setting a molecular weight standard

One method for determining molecular weight from a scattering profile is comparison to a known scattering profile with known molecular weight. This part will teach you how to set that known standard in RAW.

1. Load/use the settings from Parts 4, 5, or 6.

2. Plot all of the lysbuf2_52_001_000x.tiff files, where x is 0-9, on the main plot.

   • Tip: Section 1 Part 1 of this tutorial document teaches you how to do this.

3. Average the lysbuf2 files you just loaded. Save the average in the calibration_data folder.

4. Repeat steps 2-3 for the lys2_52_001_000x.tiff files.

5. Subtract the averaged buffer profile (lysbuf2) from the averaged sample profile (lys2).

   • Tip: Section 1 Part 1 of this tutorial document teaches you how to do this.

6. Select the subtracted profile by clicking on it. In the information panel, set the concentration in the Conc box to 4.14 (this is concentration in mg/ml).

   

7. Perform a Guinier fit on the subtracted profile.

   • Tip: Section 1 Part 2 of this tutorial document teaches you how to do this.

8. Right click on the subtracted profile and select the “Other Operations->Use as MW Standard” option.

9. Enter the molecular weight of the standard in kDa in the box that appears. For this lysozyme sample, the molecular weight is 14.3 kDa.

   

10. Click “OK” to save the molecular weight standard.

11. Save the settings for future use.