1, Dissolve polyglycerol dendrimers (PGD-G4, 0.308 g, 0.088 mmol) in 3 ml of DMSO/DMF (1:1) at 80 oC.

2. Add cholesterol chloroformate (43.4 mg, 0.097 mmol) and pyridine (0.1 ml). It will take 1–2 h to dissolve cholesterol chloroformate.

3. Stir for 1 h at 80 oC and then 9 h at 60 oC.

4. Dialyse the crude product against DMSO and then water using benzylated cellulose membrane, and then lyophilize to obtain Chol-PGDG4 as pale yellow oil.

5. Dissolve Chol-PGDG4 in 50% methanol/water (1 mM ).

Protocol 1

1. Grow bacteria in YT medium, at 37 °C 250 rpm.
2. Add 0.5M IPTG when OD600 ~ 0.8
3. Grow cells to an optical density of between 2 and 3.
4. Spin down cells.
5. Suspend in buffer A (25 mM TrisHCl pH 8.5, 0.1 mM EDTA, 5% glycerol, 1 mM DTT (dithiothreitol), 50 mM NaCl) at room temperature.
6. Lyse cells.
7. Add NaCl to 1.5 M
8. Heat 75°C for 15 min on a water bath.
9. Cool to RT in 15 min.
10. Spin and collect the supernatant.
11. Buffer exchange with buffer B (25 mM TrisHCl pH 8.5, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 100 mM NaCl).
12. FPLC on a heparin sepharose column using gradient elution with Buffer B and Buffer C (25 mM TrisHCl pH 8.5, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 600 mM NaCl).
13. Buffer exchange then load to a Q sepharose column and elute with 25 mM to 250 mM NaCl (25 mM TrisHCl pH 8.5, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 250 mM NaCl).
14. Dialyse against storage buffer (50% [v/v] glycerol, 100 mM KCl, 20 mM TrisHCl pH 8.5, 0.1 mM EDTA, 1 mM DTT, 0.25% Thesit).

Protocol 2

1. Suspend 5 g cells  in 25 ml lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, 0.1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM DTT, pH 8.0).
2. Lyse cells using ultrasound.
3, Add DNase I to a final concentration of 20 mg/ml and MgCl2 to a final concentration of 4 mM.
4. Incubate at 25°C for 30 min.
5. Heat incubation for 30 min at 72°C.
6. Spin and collect supernatant.
7. Load to a 10 ml Ni-NTA superflow column (Qiagen).
8. Wash with two volumes of buffer A (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0).
9. Elute with 100 ml of a linear gradient, starting with buffer A and ending with buffer B (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, pH 8.0).
10. Further purify by anion exchange chromatography with Q sepharose.
11. Dialyse the fraction containing the purified His-tagged Taq agaist storage buffer.

Specific activities of truncated Taq DNA polymerase
TaqWT ∼100 kU/mg
TaqΔ288 ∼77 kU/mg
TaqΔ279 ∼370 kU/mg
TaqΔ290 ∼111 kU/mg

1. Protein concentration of 1.3 mg/ml in  Tris buffer (50 mM Tris-HCl, 1 mM EDTA, 65 mM KCl, pH 7.5).

2. Dialyze protein against 1000× sodium borate at pH 8.63 (optional).

3. Dilute citraconic anhydride (11.06M, Sigma) 100-fold in DMF (N,N dimethyl formamide).

4. Make  a 2-fold dilution series of the citraconic anhydride solution in DMF. For each solution in the series, add 4 μl of diluted citraconic anhydride solution  to 400 μl protein solution (with sodium borate dialysis)
The resulting solutions containing molar ratios of citraconic anhydride to protein of approximately 80/1, 40/1, 20/1, and 10/1.

5. Incubate solutions overnight at 4° C.

6. Remove citraconic anhydride by dialysis.

Other chemicals for citraconylation.
cis- aconitic anhydride; 2,3-dimethylmaleic anhydride; exo-cis-3,6-endoxo-δ 4 -tetrahydropthalic anhydride; and 3,4,5,6-tetrahydrophthalic anhydrid

Example 1.
1. Dissolve 3 mg protein in 3 ml reaction buffer (50 mM HEPES, 300 mM KCl, 1 mM EDTA, pH8.5).
2. Add 2 µl of citraconic acid anhydride.
3. Incubate at room temperature for 1 h.
4. Dialyse with buffer S (63% [w/v] glycerol, 100 mM KCl, 20 mM TrisHCl pH 8.5, 0.1 mM EDTA, 0.5% Tween 20, 1 mM DTT).

Example 2.
1. Mix 50 ml of 27 mM citraconic anhydride (CA) in DMF with 1 liter of purified protein.
2. Adjust OD280 to 1 with buffer (50 mM Tris, pH 9.0, O.lmM EDTA, and 0.01% Tween), make sure pH is 9.0
3. Incubate for 60 minutes at 4 0C.
4. Dialyse with buffer S (63% [w/v] glycerol, 100 mM KCl, 20 mM TrisHCl pH 8.5, 0.1 mM EDTA, 0.5% Tween 20, 1 mM DTT).

glutathione
cysteine
cysteamine
n-Penthanol 1.0-10.0 mM
Lauryl Maltoside 0.06 mg/ml
n-Hexanol 0.1-10.0 mM
CETAB  0.6 mg/ml
Cyclohexanol  0.01-10.0 mM
CHAPS  10-60 mM
Tris > 0.4 M
Triton X-100 10 mM
Na2SO4 or K2SO4 0.4-0.6 M
Dodecyl Maltoside 2.0-5.0 mM
Cyclodextrin  20-100 mM
Sarkosyl 0.05-0.5

Wash beads with
1. Water 3 – column volumes
2. 0.1% SDS – 3 column volumes
3. Water – 1 column volume
4. Column Buffer – 5 column volumes