Signature Peptides of Rituximab on a bioZen Peptide XB-C18 Column Using bioZen MagBeads

LC Conditions

Column

bioZen 2.6 µm Peptide XB-C18, LC Column 50 x 2.1 mm, Ea

Brand

Biozen LC

Part No

00B-4768-AN

Phase Name

PEPTIDE XB-C18

Elution Type

Gradient

Mobile Phase

A: 0.1% Formic acid in Water

B: 0.1% Formic acid in Acetonitrile

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

85

15

2

4.5

75

25

Flow Rate

0.30 mL/min

Temperature

40°C

System

Agilent Technologies SCIEX X500B Q-TOF

Detection

LC/MS

Detection Info

Sample Notes

*Rat

Sample Preparation Details

Description

bioZen MagBeads, Streptavidin Coated - 500mg, Ea

Part No

KS0-9533

Pretreatment Note

steps 1-25 = bead activation steps 26-31 = immunocapture steps 32-42 = wash and elution

Evaporation

Evaporate to dryness

Reconstitute

beads with PBS buffer to the original slurry volume.

Steps

0

Dispense

25 µL of bead slurry into a 96 well plate

1

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

2

Add

500µL PBS Buffer to each plate well that contains beads

3

Vortex

bead slurry using mixer or pipette

4

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

5

Add

500µL PBS Buffer to each plate well that contains beads

6

Vortex

bead slurry using mixer or pipette

7

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

8

Add

500µL PBS Buffer to each plate well that contains beads

9

Vortex

bead slurry using mixer or pipette

10

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

11

Reconstitute

beads with PBS buffer to the original slurry volume.

12

Add

10 µL of biontinylated goat anti-human IgG (0.5mg/mL).

13

Transfer

plate for incubation for one hour at room temperature with a shaking speed of 1200 RPM using a deep well plate thermoshaker. Beads should stay suspended during incubation

14

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

15

Add

500µL PBS Buffer to each plate well that contains beads

16

Shake

and mix the bead slurry, using vortex or pipette

17

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

18

Add

500µL PBS Buffer to each plate well that contains beads

19

Shake

and mix the bead slurry, using vortex or pipette

20

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

21

Add

500µL PBS Buffer to each plate well that contains beads

22

Shake

and mix the bead slurry, using vortex or pipette

23

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

24

Reconstitute

beads with PBS buffer to the original slurry volume.

25

Shake

and mix slurry of anti-human IgG coated beads well, using vortex or pipette

26

Transfer

50 µL of bead slurry into each well that contains 25 µL sample. Mix well before transferring

27

Add

cover to plate using polyester plate film

28

Vortex

to mix plate

29

Transfer

plate for incubation for one hour at room temperature with a shaking speed of 1200 RPM using a deep well plate thermoshaker. Beads should stay suspended during incubation

30

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

31

Add

200 µL of PBS Buffer to each well that contains beads

32

Vortex

well and place plate on centrifuge for 2 minutes at 400RPM

33

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

34

Add

200µL of 10mM Ammonium Bicarbonate to each well that contains beads

35

Vortex

well and place plate on centrifuge for 2 minutes at 400RPM

36

Transfer

96 well plate onto magnetic stand for 30 seconds and discard liquid

37

Add

100µL of 0.1% TFA in water to each well and vortex

38

Add

pH paper to check pH value (should be lower than 3)

39

Add

plate cover and shake for 10 minutes at 1200RPM using thermomixer

40

Transfer

plate on centrifuge for 2 minutes at 400RPM

41

Transfer

96 well plate onto magnetic stand for 5 minutes and collect supernatant