Jul 21, 2025
Immuno-isolation of endogenously tagged BLTP3A^V5
- Michael Hanna1,
- Hely Rodriguez Cruz1,
- Pietro De Camilli1
- 1Yale University
Protocol Citation: Michael Hanna, Hely Rodriguez Cruz, Pietro De Camilli 2025. Immuno-isolation of endogenously tagged BLTP3A^V5. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8qyp9l2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 05, 2025
Last Modified: July 21, 2025
Protocol Integer ID: 219604
Keywords: ASAPCRN, isolate bltp3a, immuno, cell
Abstract
This protocol explains how to immuno-isolate BLTP3A (BLTP3A^V5) from endogenously edited A549 cells
Guidelines
This protocol is designed to use the following cells lines: A549 wild-type (RRID:CVCL_0023); A549 BLTP3A^V5, B6 clone (RRID:CVCL_E9W3); A549 BLTP3A^V5, C5 clone (RRID:CVCL_E9W4)
Materials
15 cm plates (Corning 353025)
10 cm plates (Corning 430293)
dPBS (Sigma-Aldrich D8537)
anti-V5 resin (ChromoTek v5tma)
Trypsin (Gibco 25300054)
Eppendorf tubes (Fischer 05-408-138)
Cell scrapper (Fisher 353086)
Magnetic rack (Fischer 12321D)
Lysis buffer:
25 mM Tris pH 7.4 (Millipore Sigma T1503)
150 mM NaCl (Millipore Sigma S5886)
1% Triton (American Bioanalytical ab02025)
Protease inhibitors (Sigma-Aldrich 05056489001)
Complete media for A549 cells:
DMEM (Gibco 11965118)
10% FBS (Gibco/LifeTech 16140-071)
Pen-Strep (Gibco/LifeTech 15140-122)
Plasmocin (Invivogen ant-mpp)
Troubleshooting
Expanding cells
From one or more 10 cm plates at ~80-90% confluency, wash once with PBS before trypsinizing cells at 37C for 3-5 mins
Collect cells lifted off of the dish using complete media into a 15 mL Falcon tube
4m
Count the number of cells per mL of media using a hemocytometer or your choice of counting method
5m
Plate 1.5E6 cells onto to two 15 cm plates per condition per replicate: A549 wild-type and BLTP3A^V5 KI, in triplicate; for a total of 12 x 15 cm plates (six wild-type and six knock-in)
10m
Let the cells grow until ~90% confluent (typically 3-4 days)
4d
Lysate and anti-V5 preparation
3h 17m
Remove media from 15 cm plate and wash with at least 10 mL dPBS and then remove
5m
At 1 mL dPBS to each 15 cm plate (it works best to work with only two plates at a time to avoid cells drying out)
1m
Using a cell scrapper, scrape up as many cells as you can
5m
Pipette up as many cells as you can using the 1 mL of dPBS added to the dish and collect 2 x 15 cm plates in a single 2 mL Eppendorf tube on ice
5m
Spin down cells at 500g for 5 min in a refrigerated (4C) table top centrifuge
5m
Remove supernatant and resuspend cells in ice cold 1 mL lysis buffer
1m
Let cells rotate at 4C for 30 mins
30m
While cells rotate, prepare anti-V5 resin: add 75 uL resin to 1 mL ice cold lysis buffer in a new 2 mL eppendorf tube, invert 3 times, place on magnetic rack; leave at 4C until ready
5m
Spin down lysate at 17000g for 10 mins in a refrigerated (4C) table top centrifuge
10m
Collect supernatant in a new, ice cold 2 mL eppendorf tube
5m
Remove lysis buffer from resin and add lysate to resin
5m
Let lysate rotate at 4C with resin for 2 hours
2h
Resin washing and storage
22m
Place tubes on magnetic rack at 4C for 1 min to capture resin
1m
Remove supernatant and replace with 1 mL ice cold lysis buffer
5m
Rotate resin for 5 min at 4C
5m
Repeat this wash one more time (total of 2 washes)
10m
After final wash, place tubes on magnetic rack at 4C for 1 min to capture resin and then remove supernatant
1m
Spin down tube at 500g for 2 mins to pellet all buffer
2m
Put tubes back on magnetic rack at 4C for 1 min to capture resin
1m
Using a 200 uL pipette, remove the last little bit of buffer from the bottom of the tubes while still on the magnetic rack
5m
Store resin in absence of buffer at -80C