简介：3X FLAG Peptide是一个合成的含有三个重复DYKXXD氨基酸序列的多肽。
纯度：>95% by LC-MS
溶解性：water 2mg/ML; TBS (5mg/ml);
20 x TBS 浓缩液
储存液配置：3xFLAG peptide溶于1X的TBS(pH7.4),浓度为5mg/ml; 分装储存于-20℃，避免反复冻融。
工作液配置：3xFLAG peptide常用工作液浓度为100ug/ml，可用于从Anti-FLAG M2亲和凝胶上洗脱3xFLAG融合蛋白。5倍柱体积的工作溶液即可充分洗脱大部分3xFLAG融合蛋白
二：FLAG-Tag Protein Purification Protocol for Mammalian Cells
General Notes The following protocol is based on and optimized for over expressed FLAG-tagged proteins from mammalian cells (U2OS) grown in one 10 cm2 plate transfected at 90% confluence and harvested after 48 hours. This protocol works well for co-purification of interacting proteins (based on FLAG-Ring1B/Bmi1).
1 Re-suspend and Sonicate Cells Re-suspend the frozen mammalian cell pellet in 2 mL of FLAG-Purification Suspension Buffer (1XPBS, 1mM DTT, 1X Protease Inhibitor). This re-suspension buffer does not have any lysis agents in order to preserve enzymatic activity. Purifications not harvesting for activity may use actual lysis buffersin addition to sonication (1% Triton X or other). This protocol uses sonication only.
Transfer the mixture to a clean 2 mL microtube and sonicate 2 times for 10 continuous seconds each, with 1 min pause in between using “Amplitude Setting 1 [out of 20]” on a Misonix XL- 2000 sonicator.
2 Prepare and Incubate Cell Lysate with Beads Centrifuge the cell lysate for 20 min at 12,000 g at 4o C to pellet lysate debris.
Use a 2 mL “micro” purification column and flush the column with 2mL of the FLAG-Purification Suspension Buffer.
Transfer 100 µL of M2 FLAG Affinity beads to the column and wash the gel away with 2 mL of FLAG-Purification Suspension Buffer.
Dry the nose (bottom) of the column and close it with the cap very tightly, then transfer the supernatants from the centrifuged cell lysate to the column gently avoiding the debris pellet.
Cap the column and incubate on a rotator at 4oC for 1.5 hours. This may be extended to overnight if enzymatic activity of protein is not a concern.
3 Wash The Column Wash the column after incubation with at least 10 mL of FLAG-Purification Suspension Buffer. If the column doesn’t flush by gravitational flow, centrifuge at low speed (~300-400 RPM) for just a few seconds.
4 Elution To lute the column by incubating the beads at 4 o C for 30 min with 100 µL of 3X FLAG Peptide at a 100 ng/mL working concentration [the “3X” in 3X FLAG Peptide is not any indication of concentration, it simply refers to 3 tandem repeats of FLAG sequence in the peptide]. You can make a 1 mL of the working concentration by mixing 20 µL of the commercially available 5 mg/mL stock with 980 µL of TBS.
At the end of the incubation centrifuge at low speed for a few seconds if column does not elute by gravitational flow. You may do additional elution steps to recover more protein.
三：Purification of Halo or FLAG tagged protein complexes from transiently transfected HEK293 cells for MudPIT mass spectrometry
1. Seed a 15 cm dish with ~2 x 107 cells. Allow cells to recover overnight.
2. Transfect with 7.5 µg of DNA expressing FLAG or Halo tagged protein of interest.
3. Harvest cells ~48 hours post transfection:
a. aspirate media,
b. wash with 10-20 ml ice-cold PBS,
c. add 10 ml ice-cold PBS and remove cells with a cell scraper and transfer cells to a 15 ml tube on ice,
d. spin at 2000 x g for 10 minutes at 4°C,
e. remove PBS; resuspend in ~1 ml ice-cold PBS and transfer to 1.5 ml microfuge tube.
f. spin at 2000 x g for 10 minutes at 4°C,
g. Freeze pellets in liquid nitrogen and transfer to -80°C (protocol can be stopped at this point).
4. Thaw cells on ice and resuspend in 300 µl mammalian lysis buffer containing protease inhibitors.
5. Pass through a 26G needle using a 1 ml syringe 5-10 times.
6. Spin at max speed 10 minutes at 4°C.
7. Transfer supernatant to a clean tube (protocol can be stopped at this point; freeze pellets in liquid nitrogen and transfer to -80°C).
8. Add 700 µl ice-cold PBS.
9. Spin at max speed 10 minutes at 4°C.
10. Transfer supernatant to a clean tube containing washed anti-FLAG agarose or HaloLINK resin. Leave final 10-20 µl to avoid disturbing pellet.
11. Incubate with mixing for 1 hour at 4°C.
12. Wash beads four times (spin at 2000 x g for 1 minute at 4°C, discard supernatant, add 1 ml ice-cold wash buffer and repeat).
a. FLAG elution: Add 100 µl FLAG elution buffer and incubate 30 minutes at 4°C with shaking.
b. Halo elution: Add 100 µl Halo elution buffer and incubate 1-2 hours at 25°C with shaking.
14. Spin at 2000 x g for 1 minute. Transfer the 100 µl supernatants to an empty micro bio spin column (BioRad #732-6204) inserted into a 1.5 ml microfuge tube. Spin at 2000 x g for 1 minute.
15. Aliquot 20 µl of the eluate for analysis by SDS PAGE. Digest the remaining 80 µl eluate for analysis by MudPIT mass spectrometry.
Preparation of anti-FLAG agarose or HaloLINK resin
1. Vortex container of anti-FLAG agarose (sigma A220) or HaloLINK resin (Promega G1912) to resuspend.
2. Aliquot 100 µl slurry per purification into 1.5 ml microfuge tubes.
3. Wash 3 times with wash buffer (spin at 2000 x g for 1 minute at 4°C, remove supernatant, add 1 ml ice-cold wash buffer and repeat). Only remove final wash just prior to adding lysate.