Checking anti-LaORF1 and anti-LaG94 beads with or without XL
Date: 06/30/20
Beads:
1) anti-LaORF1-5 (nanobody beads, 08/01/19)
2) anti-LaORF1-5 (nanobody beads, 10/24/19
3) anti-LaG94-10 (nanobody beads, 10/24/19)
XL nanobody to Epoxy beads after conjugation
Take the following aliquots of beads:
2x 5ul of anti-LaORF1-5 (08/01/19)
3x 5ul of anti-LaORF1-5 (10/24/19)
1x 20ul of anti-LaORF1-5 (10/24/19)
2x 5ul of anti-LaG94-10 (nanobody against GFP, 10/24/19) beads
1x 20ul of anti-LaG94-10 (10/24/19)
Wash once in 1ml of 1xPBS
Resuspend beads in 400ul of 20mM DMP in Borate buffer (made fresh)
We should use 2ml per reaction with 100ul of beads. When we used LaORF1 beads in anti-ORF1 IP, 2nd batch of beads made on 10/24/19 had significant LaORF1 leakage even after XL. This time, I was trying to see if using larger volume or higher concentration of DMP can improve XL efficiency.
1) anti-LaORF1 (08/01/19)_5ul_400ul of 20mM DMP
2) anti-LaORF1 (10/24/19)_5ul_400ul of 20mM DMP
3) anti-LaORF1 (10/24/19)_5ul_400ul of 40mM DMP; check 5ul out 20ul on gel
4) anti-LaORF1 (10/24/19)_20ul_400ul of 20mM DMP
5) anti-LaG94 (10/24/19)_5ul_400ul of 20mM DMP
6) anti-LaG94 (10/24/19)_20ul_400ul of 20mM DMP; check 5ul out 20ul on gel
Perform XL reaction @ RT on the wheel for 30’.
After 30’, remove the XL solution
Wash beads with 1ml of PBST
Wash beads again with 1ml of TBST for 5’ @ RT
Wash beads again with 1ml of PBST
Wash beads with 1ml of 1x PBS
Elute the beads with 10ul of LDS, 70 °C for 5’ with mixing
Collect the eluate
Add DTT to 50mM to all samples and heat @70 °C for 10’
Run 4-12% Bis-Tris gel to analyze the elution, Sypro stain
1) Unstained Marker_1ul
2) LaORF1 (08/01/19)_5ul_no XL
3) LaORF1 (08/01/19)_5ul_20mM DMP
4) LaORF1 (10/24/19)_5ul_no XL
5) LaORF1 (10/24/19)_5ul_20mM DMP
6) LaORF1 (10/24/19)_5ul_40mM DMP
7) LaORF1 (10/24/19)_5 out of 20ul_ 20mM DMP
8) LaG94 (10/24/19)_5ul_no XL
9) LaG94 (10/24/19)_5ul_20mM DMP
10) LaG94 (10/24/19)_5 out of 20ul_20mM DMP
11) BSA_25ng
12) BSA_100ng
13) Prestained Marker_5ul (not shown)
2 second exposure
Conclusion: XL worked for all beads tested. The 2nd batch of LaORF1 beads had more nanobody coupled to beads and nanobody leakage was a bigger problem even after DMP XL. The nanobody or antibody leakage problem and the effect of DMP XL seem to be antibody dependent and may vary among batches of the same nanobody (or antibody). Using 2ml of 20mM DMP per 100ul of beads is fine. No need to increase the volume of DMP concentration.
Note: The loading buffer seemed to be contaminated. Make fresh 1.1x LDS.