Chlamy flagella rafts labelling protocols
PURPOSE:
To label flagellar rafts containing glycoproteins with floresecent microspheres and track their movements with nanometer precision microscopy
To show relation of bead movement and raft motility(flagellar surface motility) with IFT (intraflagellar transport)
To observe and measure steps taken by beads with fast imaging techniques
PROTOCOLS:
Covalenly Coating Beads with Antibody: Antibody-Bead coating protocol for 100nm diameter beads
Wash carboxy-modified 0.1 um microspheres in activation buffer [start with 100ul of beads + 300ul of activation buffer] [Activation buffer: 10 mM MES, 100mM NaCL, pH 6.0 ]
Centrifuge at 12700 rcf (15000rpm) at 4C, for 30 min
Take out supernanatant
Resuspend in 400 ml of activation buffer and sonicate for 30 sec
Centrifuge at 12700 rcf (15000rpm) at 4C, for 30 min, remove supernatant and resuspend in 400 ul of activation buffer, vortex, and sonicate for 30 sec
Add 5mg EDC (EDAC) and 5 mg s-NHS (NHS-S), make sure crosslinkers are fresh and frozen in -20C. Sonicate for 30 sec and shake solution at least for 20 min at RT
Add 1.4 ul 14.3M BME
Wash 2 times in coupling buffer (in first step buffer and ) [Coupling buffer: 100mM PO4 buffer, pH 7.4,(prepare by adding 77.4 ml 1M Na2HPO4 and 22.6 ml 1M NaH2PO4)]
Add 400 coupling buffer, total will be 800 ul, Spin solution at 12700 rcf (15000rpm) at 4C, for 30 min, remove supernatant and resuspend in 400 ul of coupling buffer, vortex, and sonicate for 30 sec, make sure the beads are well suspended
Repeat centrifugation at 12700 rcf (15000rpm) at 4C, for 30 min, remove supernatant and for final resuspention, use 200ul of PBS, total volume should be 200ul , vortex, and sonicate for 30 sec, make sure the beads are well suspended
Add 20ul of FMG-1 antibody and 50 ul of 100 mg/ml BSA [FMG-1 antibody first!, then BSA], vortex, and sonicate for 30 sec, inubate for 1 hours at RT with constant mixing (or O/N at 4C)
Complete up to 1 ml by adding 800 ul of PBS
Spin solution at 12700 rcf (15000rpm) at 4C, for 30 min, remove supernatant
Resuspend in 200 ul quenching solution [Quenching solution: 30mM hydroxylamine hydrochloride (NH2OH.HCl) in PBS, pH 8.0, [dissolve 0.42 g NH2OH.HCl in 200 ml PBS and adjust pH] ]
Shake at RT for 30 min
Rinse the beads once in PBS buffer. Resuspend in the original volume of 100ul PBS [We added 400ul instead of 100 ul, it results in 4X dilution]
Add 0.1 % azide (NaN3, prevents growth of bacteria, 100% corresponds to 1 g/ml, use 10 % azide stock solution, e.g. add 1 g sodium azide to 10 ml of dH2O) [I added 4 ul for 400ul solution]
Store at 4C (Never freeze beads!)
Chlamy Growth (M1)
Streak cells on TAP agar medium, let them grow continuous light for a week
Pour 2 ml of M1 medium into glass culture
Take some chlamy colony with a pipette tip to grow chlamy in MI medium
Resuspend all cells via pipetting
Incubate chlamy at rotator for at least 2 days to get longer flagella, healthy, and synchronous culture (one day is also ok) under continuous light, 21C
Slide Preparation
Dilute antibody coated beads by adding 1 ul to 10ul (it approximately results in 10X dilution) and sonicate for 10 sec before use
Prepare 100X diluted CaCl2 by using 1M CaCl2 [1 ul of 1M CaCl2+99 ul of dH2O]
Keep beads on ice while you are using [store beads at 4C for further usage, sonicate for 15 sec before use if it is not fresh]
Check whether chlamy has flagella and healthy or not, under light microscopy
Take 100 ul of culture and centrifuge for 30 sec at 0.5 rcf or 2.3 rpm, [if pellet is not as much as desired, start with more volume like 200 ul and remove 180 ul to get 20 ul pellet]
Remove 80 ul of supernatant
Dilute the cells and media by adding 80 ul of dH2O (5X dilution)
Take 10 ul of 5X diluted cells into small centrifuge tubes
Add 1 ul of 10X diluted antibody coated 100 nm beads into 10 ul of cells, mix gently
Incubate on ice at least 5 min –this is actually time passing during you treat cover glass and slide with casein-
Treat coverglass with 1ul of poylysine, spread
Air dry for 1 min
Prepare chamber by using double sided tape
Add 1 ul of 100X diluted CaCl2 in 10ul cell-bead suspension, mix gently [Final dilution of CaCl2 will be 1000X, 1 mM CaCl2]
Flow 10 ul of cell-beads suspension into the chamber, and seal with nail polish
Keep slide up side down until microscopy