Hybridoma Protocol

 Growing hybridoma and collecting supernatant: o Prepare hybridoma medium. For TROMA-I, the suggested medium was: 400 ml IMDM 100 ml FBS 0.5 ml 50 mg/ml gentamicin **I usually supplemented this with Pen/Step to be safe – roller bottles get contaminated very easily.** o Thaw cells in 37C water bath. As soon as cells are thawed, spray tube with ethanol (water baths are dirty). Transfer cells to a 15 ml conical containing hybridoma medium and spin down. Do all of this quickly – freezing medium contains DMSO which makes the cells unhappy. o Remove medium and resuspend pellet in 10 ml fresh hybridoma medium. Transfer cells to 75 cm tissue culture flask. Check cells under microscope to make sure they look okay, and culture in incubator. o Once they are growing well, you will probably need to split the cells about 1:2 or 1:4 every other day, but you should check on them every day. At this point, you don’t want to let the media get too yellow or the cells to get sick, because they will stop producing antibody. To split the cells, remove all cells and media from the flask. Hybridoma cells can be slightly adherent, so you might need to wash the flask a bit, but you won’t need to trypsinize or scrape the cells. Spin down the cells, remove, filter, and freeze the supernatant, resuspend the cells in appropriate volume of fresh media, and transfer to new flasks. o Once you have expanded the cells enough to have 2-3 large flasks (about 40 ml cultures), you should freeze cells. To do this, spin down 10^7 cells and resuspend in 0.5 ml freezing media (can use media with a high amount of serum +10% DMSO, or straight serum +10% DMSO). Transfer to cryovial and immediately place in -80C freezer. The next day, transfer the cryovials to liquid nitrogen for long term storage. o Continue expanding cells. Once you have about 4 large flasks that are ready to be split, you can start a roller bottle. Split one of these flasks into 2-4 large flasks to continue growing normally. Spin down the contents of the other 3 flasks, filter and freeze the supernatant, and transfer the cells to a roller bottle containing 400 ml medium. o Tighten the lid all the way, then gently place into roller apparatus, trying not to let any media touch the inside of the lid. Once the bottle is in the apparatus, loosen the lid, and turn on the machine. Allow cells to grow until media turns very yellow. The cells will die, and this is okay. o Collect supernatant by spinning down cells in the plastic bottles we normally use for bacterial cultures. Filter the supernatant and freeze.  Purifying antibody from supernatant:  This protocol is for Protein G purification. Check your antibody isotype to make sure Protein G is the best option for you.  Setting up and running column: o Make sure the bottom of the column is capped. Pipet 0.5-1 ml Protein G-Sepharose beads into column (the amount of Protein G to use depends on the amount of supernatant you have; also a column with a wider diameter will run much faster than the smaller ones). Add 150 mM PBS, pH 7.4 to fill column ~ 2/3. o Balance column by passing 150 mL 150 mM PBS, pH 7.4 through column at 4 C. o Pass supernatant through column 3x at 4 C. Each time, collect supernatant in a clean bottle and reuse. It is okay to leave this running overnight, because when the top bottle of supernatant is empty, the column should stop flowing. o Wash with 2 L 150 mM PBS pH 7.4 (overnight). Continue washes until OD280 <0.002. Cap the bottom of the column and detach from tubing.  Elution: o Bring column to your bench and suspend with clampy-apparatus-thing. Remove cap and allow all PBS to run out of the column. To elute, recap tube, add 1.5 V (I actually usually did 2 ml) 0.1 M glycine, pH 2.4, and allow to sit briefly. Collect 0.5 ml fractions. You can either collect all eluate, or check the pH while eluting and wait until pH drops before collecting fractions. o Measure the protein concentration in each fraction using a Bradford assay. In Eppendorf tubes, combine 400 ul ddH2O, 100 ul Bradford dye, and 10 ul sample. Mix well, and use the Protein Concentration protocol on the spectrophotometer to measure protein concentration. Select “weiC” for your standard curve. o You should be able to identify a peak in protein concentration in your fractions. Select the peak fractions and combine them. o To regenerate column, run 3 cycles of high pH and then low pH solution . High pH solution (8.5): Tris 1.2114, NaCl 2.9 -> 100 ml . Low pH solution (4.5): NaCOOCH 0.8203, NaCl 2.9 -> 100 ml  Dialysis: o Boil a piece of dialysis tubing for 5 minutes. Clip one end of the tubing, pipet your combined eluted fractions into the tube, and clip the other end of the tubing. Be very cautious, as the clips sometimes come undone during dialysis. You might want to wrap them with tape to be safe. o Dialyze overnight in 2000 mL 150 mM PBS, pH 7.4 at 4C, stirring slowly. Change the dialysis buffer once overnight.  Checking antibody concentration and purity: o Carefully unclip tubing and remove purified antibody. Use a Bradford Assay to determine protein concentration. o Boil 10 ul of purified antibody and 10 ul of hybridoma supernatant in 2x sample buffer for 5 minutes, and run on a 10% SDS-PAGE gel: . Separating gel:  5 ml Protogel  7.5 ml 2x separating buffer  2.5 ml ddH2O  45 ul 10% APS  10 ul Temed . Stacking gel:  1 ml Protogel  5 ml 2x stacking buffer  4 ml ddH2O  75 ul 10% APS  15 ul Temed o Coomassie stain gel: . Rock 30 minutes in Coomassie stain. . Wash 2-3x with H2O. . Destain for 2 hours in 10% methanol/10% acetic acid (cover in saran wrap). o Dry gel using machine in Zhuang lab.