Concentrating and buffer exchange of anti-ORF1 (4H1)
Date: 09/02/21
New batch of anti-ORF1 from Abmart (Project ID 42298-1, shipped 08/20/21, received 09/01/21) @ 0.7mg/ml, 18mg total.
Storage buffer: 100mM Glycine, 50mM Tris; however, no proclin 300 was added this time.
The antibody does not look completely transparent. There is some precipitation at the bottom of the glass bottle containing the antibody.
Take 2x 700ul (~0.49mg), spin @ 14k rpm @ 4ºC for 5’
Observe a tiny white pellet at the bottom of each tube
Tube 1: transfer 350ul supernatant to another tube (1b); add 350ul of 4M AmSO4 to 1a (original tube) and 1b
• 1a and 1b: 350ul of antibody and 350ul of 4M AmSO4, 700ul total, mix well; did not see precipitation immediately.
• Control tube: 350ul of 1x PBS and 350ul of 4M AmSO4
Tube 2: 700ul of antibody
Put tubes 1a, 1b, 2 and control tube all in speed vac to reduce the volume
• Tube 1a and 1b: stop when start to see antibody precipitation in these two tubes but not in the control tube (antibody should precipitate out when AmSO4 is 2-3M; AmSO4 should not precipitate out even @ 4M)
• Tube 2: reduce the volume to half (350ul) then buffer exchange the antibody into 0.1M NaPhosphate, pH7.4
Check the volume in the control tube in 10’ interval
Time 0: 700ul in each tube
After 10’: ~600ul
After 20’: ~500ul
After 30’: ~400ul (Take tubes 1a and 1b out)
After 40’: ~300ul
Add another 5’: final volume in tube 2 is 250ul
Tubes 1a and 1b:
• Spin @ 14k rpm @ 4ºC for 5’
• Resuspend each in 125ul of 0.1M NaPhosphate, pH7.4, mix thoroughly by pipetting and vortexing
• Combine sample in both tubes, total 250ul
• Spin @ 14k rpm @ 4ºC for 5’
• Transfer the supernatant to a fresh tube (There is a tiny pellet left in the tube)
Tubes 2:
• Spin @ 14k rpm @ 4ºC for 5’
• Transfer the supernatant to a fresh tube (The tiny pellet left in the tube observed before putting the tube in speedvac is still there)
• Equilibrate two 0.5ml Zeba 7K desalting columns (capacity upto 130ul sample) with 0.1M NaPhosphate, pH7.4
• Transfer 125ul of supernatant to each Zeba 7K desalting column
• Recovered antibody (in 0.1M NaPhosphate, pH7.4); did not lose any volume (250ul final)
Check the antibody before and after AmSO4 precipitation or buffer exchange by gel Marker
Calculate Volume change of AmSO4 precipitated or desalted antibody
Volume change from 700ul to 250ul (2.8x fold)
Take 1ul of each sample, add 24ul of 1.1x LDS and 3ul 0.5M DTT (total volume 28ul); load 10ul on gel
Take 1ul of original antibody, add 8ul of 1.1x LDS and 1ul 0.5M DTT (total volume 18ul); load 10ul
Heat samples @ 70ºC for 10’
Run 4-12% Bis-Tris gel
1) Marker
2) Original antibody
3) AmSO4 precipitated antibody
4) Desalted antibody
5) BSA_50ng
6) BSA_150ng
Check antibody concentration by BCA assay
• Original antibi: 0.7mg/ml
• AmSO4 precipited antibody: 1.2mg/ml (volume reduced from 700ul to 250ul; antibody recovery (1.2ug/ul*250ul)/(0.7ug/ul*700ul)->61%)
• Desalted antibody: 1.4mg/ml (volume reduced from 700ul to 250ul; antibody recovery (1.4ug/ul*250ul)/(0.7ug/ul*700ul)->71%)
