Removing 3xFLAG by Zeba desalting columns
Date: 08/13/20
Prepare mock sample
Take 20ul 1mg/ml 3xFLAG peptide in extraction buffer to each tube (1mg/ml 3x FLAG peptide was diluted from 5mg/ml stock in 500mM extraction buffer*)
- 20mM HEPES, pH 7.4, 500mM NaCl, 1% Triton X-100 (v/v)
Add 10ul of in 500mM extraction buffer
Pool these two fractions together (30ul total)
Save 10ul 3xFLAG elution for gel analysis
Split the rest into 2x 10ul aliquot pass through two different kinds of Zeba desalting columns
Removing 3x FLAG using Zeba columns
Take one 75ul Zeba 7K column and one 75ul Zeba 40K column
Remove the storage buffer from Zeba columns, spin @ 1000g for 1’
Equilibrate Zeba columns with 3x 50ul of 20mM HEPES, pH 7.4, 250mM NaCl, 1% Triton X-100 (v/v), spin @ 1000g for 1’
- Sample volume range 5-14ul; no need to add stacker when sample volume >5ul
Sample recovery: load 10ul of sample on each spin column and spin @ 1000g for 2’
Collect the desalted samples for gel analysis
The concentration of 3xFLAG peptide should be 0.67mg/ml (20ul of 1mg/ml 3xFLAG mixed with 10ul of buffer)
Dilute BSA standard first from 2mg/ml to 0.67mg/ml (same concentration as 3XFLAG) and then make 1:10 dilution; load 1, 2, 5 and 10ul of 0.067mg/ml BSA standard; This will be equivalent to 10, 20, 50 and 100% of 0.67mg/ml BSA
Run 1ul of 3xFLAG peptide before and after desalting on gel with BSA standards (15-well 4-12% Bis-Tris gel); Run the gel @ 200v for 25’
1) Marker
2) 3xFLAG_before desalting-1ul (670ng)
3) 3xFLAG_Zeba 7K_1ul
4) 3xFLAG_Zeba 40K_1ul
5) BSA_1ul
6) BSA_2ul
7) BSA_5ul
8) BSA-10ul (670ng)
Testing BioRad P30 spin column
Date: 08/14/20
Starting material: same mock sample from the day before (same dilution from the same aliquot of 1mg/ml 3xFLAG)
Removing 3x FLAG using BioRad P30 spin columns
Take two BiorRad P30 spin columns
Remove the storage buffer columns, spin @ 1000g for 2’
Equilibrate P30 columns with 3x 500ul of 20mM HEPES, pH 7.4, 250mM NaCl, 1% Triton X-100 (v/v), spin @ 1000g for 2’ (manufacturer’s recommendation is 1’; 1’ is enough to remove all storage buffer)
Load 20ul of 0.67mg/ml of 3x FLAG on one P30 spin column and load 75ul on the other one (Sample volume range of P30 column is 20-75ul)
Sample recovery: Spin @ 1000g for 2’ (manufacturer’s recommendation is 4’)
Collect the desalted samples for gel analysis
Recovered the sample volume was larger than what was loaded on the column
• Collected 30ul from the column loaded 20ul (“20ul-1”; 1.5x original volume)
• Collected 90ul from the column loaded 75ul (“75ul-1”; 1.2x original volume)
Spin the P30 tubes for another 2’ @ 1000g (total 40ul) collect flow-through
• Recovered another 6ul from each column (“20ul-2” and “75ul-2”)
The concentration of 3xFLAG peptide should be 0.67mg/ml (20ul of 1mg/ml 3xFLAG mixed with 10ul of buffer)
Prepare BSA standard as described above
Analysis desalted 3xFLAG peptide on gel with BSA standards (15-well 4-12% Bis-Tris gel);
Run the gel @ 200v for 25’
1) Marker
2) 3xFLAG_before desalting-1ul (670ng)
3) 3xFLAG_Zeba 7K_1ul
4) 3xFLAG_Zeba 40K_1ul
5) 3xFLAG_P30 20ul-1_1.5ul
6) 3xFLAG_P30 20ul-2_1.5ul
7) 3xFLAG_P30 75ul-1_1.2ul
8) 3xFLAG_P30 75ul-2_1.2ul
9) BSA_1ul
10) BSA_2ul
11) BSA_5ul
12) BSA-10ul (670ng)
I left the gel in Acquastain over the weekend. The 3xFLAG ran to the bottom of the gel might have diffused out over time. Will re-run this set of samples.
Removing 3x FLAG using Zeba 7K, 40K and BioRad P30 spin columns
Date: 08/20/20
Remove the storage buffer columns (see above for details for each column)
Equilibrate columns with 20mM HEPES, pH 7.4, 250mM NaCl, NO DETERGENT (buffer exchange)
Mock sample: 0.67mg/ml of 3x FLAG plug 0.67mg/ml BSA (mixture of 3XFLAG and BSA at the same concentration: two parts of 1mg/ml 3xFLAG with 1 part of 2mg/ml BSA)
Load 20ul on P30; 12ul on Zeba 7K or 40K
Recover samples by centrifugation
Volume recovered:
Zeba 7K: 12ul (same as initial volume loaded on the column, 1x); run 1ul on gel
Zeba 40K: 18ul (1.5x of initial volume); run 1.5ul on gel
P30: 1st 2’ spin, 34ul
P30: 2nd 2’ spin, 6ul (total volume recovered from both spins: 34+6=40ul; 2x of the initial volumn); run 2ul of sample recovered from each spin
Re-run samples from 08/14/20 together with samples from this experiment
Run the gel @ 200v for 30’
1) Marker
2) 3xFLAG_before desalting-1ul (670ng)_081420
3) 3xFLAG_Zeba 7K_1ul_081420
4) 3xFLAG_Zeba 40K_1ul_081420
5) 3xFLAG_P30 20ul-1_1.5ul_081420
6) 3xFLAG_P30 20ul-2_1.5ul_081420
7) 3xFLAG_P30 75ul-1_1.2ul_081420
8) 3xFLAG_P30 75ul-2_1.2ul_081420
9) 3xFLAG/BSA_before desalting_1ul (134ng)
10) 3xFLAG/BSA_before desalting_1ul (335ng)
11) 3xFLAG/BSA_before desalting_1ul (670ng)
12) 3xFLAG/BSA _Zeba 7K_1ul
13) 3xFLAG/BSA _Zeba 40K_1.5ul
14) 3xFLAG/BSA _P30 1st_2ul
15) 3xFLAG/BSA _ P30 2nd_2ul
Fix the gel with 40% Methanol, 7% HAc for 45’
Wash the gel with dH2O for 10’
Wash the gel with 0.1M HCl for 10’
Wash the gel with dH2O for 10’
Blue sliver stain for 2hr
Destain the gel and take a picture
Let the gel destain overnight. The stain of the 3xFLAG peptide disappeared overnight.
Notes: 3x FLAG peptide will diffuse from the gel over time. Before staining, better fix the gel. Even with fixing, the stain of 3xFLAG peptide may get weaker or lost overnight