See attached below;
Determination of molecular weight of Keratin using CE-SDS MW protein kit (Beckman Coulter)
1) Preparation of protein standard/sample 1.1 Protein Standard; the protein size standard (10-225 kDa) was prepared following the PA800 plus application guide.
- Remove protein size standard from fridge, leave it at room temperature for 15 min. - Mix standard and pipet 5 uL of size standard into a micro-centrifuge tube. - Add 42.5 uL of sample buffer (containing SDS) into the micro-centrifuge tube. - Add 1 uL of internal standard protein (10 kDa) into the micro-centrifuge tube. - Add 2.5 uL of mercaptoethanol, cap tube tightly and mix thoroughly. - Heat the mixture in heating block at 95-100oC for three minutes. - Cool the tube before injection. The protein size standard is good for 24 hours. The protein size standard was mixed with SDS sample buffer at an elevated temperature. The reaction should produce SDS-protein complexes having similar charge densities. When standard was electrophoretically separated, their mobilities would depend on their sizes. Smaller proteins have greater mobilities.
1.2 Protein sample - Dilute protein sample with the SDS-MW sample buffer for a total 95 uL volume to give a final protein concentration range of 0.2 – 2.0 mg/mL. - Add 2 uL of internal standard (10 kDa) into the micro-centrifuge tube. - Add 5 uL of mercaptoethanol, cap the tube tightly and mix thoroughly. - Heat the mixture in a heating block at 95-100oC for three minutes. - Cool the tube before injection. The protein sample is good for 24 hours.
2) CE instrumentation setup • Installing capillary column A 50 um ID bare fused silica column was used for separation, Total length 34 cm, effective length 24 cm (the shortest length that can put in a cartridge). Flush the column with 1M NaOH, HCl, DI water for 30 min (each of those solutions) for new capillary column. • Method setup for HP3D CE Column: Bare-silica column 50 um ID, 360 um OD, L 34/24 cm
Preconditioning Event Duration Comments Flush - 0.1 M NaOH 3 min 0.1 M NaOH rinse to clean capillary surface Flush - 0.1 M HCl 3 min 0.1 M HCl rinse to neatralize capillary surface Flush - DIW 3 min Water rinse to remove the acid residue Flush - SDS buffer gel 15 min SDS gel rinse to fill the capillary with SDS gel
Post conditioning Flush - DIW 10 min Remove the used gel inside the capillary tube
Injection: Negative polarity -5kV, 30 second Voltage: Negative polarity -18 kV Column temperature: 25oC
Detection: 220 nm or 220 nm with reference at 350 nm.
Note: SDS gel buffer can be used to multiple runs.
