Checking SLE Western samples from Seattle
Date: 04/16/21
11 samples in SDS sample buffer
The SDS sample buffer is 1x Laemmli containing 50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% beta-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue.
1) SLE1073 20200820
2) SLE1237 20191007
3) SLE1284 20191211
4) SLE1337 20200923
5) HC57 20201215
6) HC2229 20200728
7) HC2431 20200903
8) RA1300 20201210;
9) RA1339 20201001
10) RA1345 20201214
11) RA1346 20210120;
Positive control for Western
N2102Ep clarified whole cell lysate in extraction buffer: 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100; total protein concentration 10.18mg/ml
25ug of N2102 lysate will give us a clear Orf1p signal by Western Wiki entry 12/19/19 - PA-1, N2102Ep and NTERA2 anti-ORF1 IP
Make 1:10 dilution of N2102Ep lysate in 1x LDS with 50mM DTT (final concentration 1ug/ul)
Sample reduction before gel loading, 70°C for 10’
Load different amounts of samples with N2102Ep and BSA standard
Gel 1: 0.5ul of each Western samples
1) Marker
2) SLE1073 20200820
3) SLE1237 20191007
4) SLE1284 20191211
5) SLE1337 20200923
6) HC57 20201215
7) HC2229 20200728
8) HC2431 20200903
9) RA1300 20201210;
10) RA1339 20201001
11) RA1345 20201214
12) RA1346 20210120
13) N21202Ep_1ug/ul_1ul (1ug)
14) N21202Ep_1ug/ul_5ul (5ug)
15) BSA_200ng
Gel 2: 15ul of 3 SLE samples (Did not use SLE1073 20200820 because its sample volume is significantly less than the other 3)
Take 2 x 15ul aliquots from each sample; one load directly after reducing; the other one was put in the speed vac to reduce the sample volume to less than 5ul first, then add 9ul of 1.1x LDS and 1ul of 500mM DTT; reduced @ 70°C for 10’ before loading
Gel 2: 15ul of SLE samples with N2102Ep control
1). Marker
2) SLE1237 20191007
3) SLE1284 20191211
4) SLE1337 20200923
5) Space
6) Marker
7) SLE1237 20191007_LDS
8) SLE1284 20191211_LDS
9) SLE1337 20200923_LDS
10) N21202Ep_1ug/ul_1ul (1ug)
11) N21202Ep_1ug/ul_3ul (3ug)
12) N21202Ep_1ug/ul_5ul (5ug)
13) BSA_50ng
14) BSA_150ng
15) Marker
4/21/21
Take 3x 45ul of RA1346 20210120; treat them differently before loading
1. Heat the sample directly @ 70°C for 10’; then put the tube in speed vac, reduce sample volume to 10ul
2. Put 45ul of sample in speed vac for 20’, reduce volume to 10ul; add 1ul of 0.5M DTT; heat @ 70°C for 10’
3. Put 45ul of sample in speed vac for 20’, @ 70°C for 10’; add 1ul of 0.5M DTT and 1ul 4x LDS; heat @ 70°C for 10’
Loading: 1) Marker
2) Space
3) RA1346_direct
4) RA1346_DTT
5) RA1346_DTT_LDS
6) Space
7) N21202Ep_5ug
8) N21202Ep_10ug
9) N21202Ep_20ug
10) Space
11) BSA_50ng
12) BSA_150ng
Anti-ORF1 Western Heat the sample directly @ 70°C for 10’; then put the tube in speed vac, reduce sample volume to 10ul
Run 15-well 4-12% Bis-Tris gel
1) Marker
2) SLE1073 20200820
3) SLE1237 20191007
4) SLE1284 20191211
5) SLE1337 20200923
6) HC57 20201215
7) HC2229 20200728
8) HC2431 20200903
9) RA1300 20201210;
10) RA1339 20201001
11) RA1345 20201214
12) RA1346 20210120
13) N21202Ep_5ug
14) Marker
15) N21202Ep_20ug
Wet transfer, 70V for 1.5hr
Stain the membrane with Ponceau and get a light image for loading
Wash the membrane in TBST to get rid of Ponceau
Ponceau stain was weak
Block both blots with TBST/5% milk @ RT for 2hr
Primary Ab: Mouse anti-ORF1 antibody, 1:1,000, 4°C overnight (2ug/ml)
3 quick rinse with TBST followed by wash with TBST, 3x 10’ @ RT
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
3 quick rinse with TBST followed by wash with TBST, 3x 10’ @ RT
5 minute exposure, super sensitivity, auto tone
Re-probing
Wash the membrane in TBST, RT 10’ to remove the chemiluminescent substrate
Strip the membrane with Restore™ PLUS Western Blot Stripping Buffer (Thermo Fisher, cat # 46430); RT for 10’
Wash the membrane in TBST, RT 10’
Block both blots with TBST/5% milk @ RT for 2hr
Primary Ab: Mouse anti-ORF1 antibody, 1:500, , RT 1hr
3 quick rinse with TBST followed by wash with TBST, 3x 10’ @ RT
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
3 quick rinse with TBST followed by wash with TBST, 3x 10’ @ RT
15 minute exposure, super sensitivity, auto tone
