Protocols

Following are the labeling protocols used by David Knowles working as a PostDoc in the laboratory of Mohandas Narla.



Magic Solution

Antioxident - reduces the photo cross-linking effect but it reduces fluorescence yeild

1mM NaH3 (Sodium Azide)  0.003g/50ml
5mM Trolox,     0.062g/50ml
   250.29g/Mole, Aldrich Chem Co P.O.Box 355


10x PBS

Into 1 litre

80g NaCl
2g KCl
14.4g Na2HPO4
2.4g KH2PO4


1x PBS
100ml 10xPBS + 900ml H20 + Bovine Serum Albumin (BSA) 0.05g%


FTSC & ETSC Labeling Glycophorin A

FTSC - fluorescein-5-thiosemicarbazide, Mol Probes F-121, 421.4g/mole, e=85,000, solvent pH 9.
ETSC - eosin-5-thiosemicarbazide, Mol Probes E-120 (Discontinued Product)

FTSC labels oxidized sialic acid residues on glycophorin A. Mild oxidation is produced by NaIO4 at a final concentration of 0.5mM.

NaIO4 213.9g/mole. Make a 10mM Stock = 21mg/10ml. Add 50ul of stock per 1ml of buffer with 10ul packed RBC's and incubate for 12min at 0C in the dark. Wash 3x. then

FTSC Stock measured by spec. incubate in FTSC for 30min at 50ug/ml


FMA Labeling Band 3

Molecular Probes F-150 MW 427g/Mole, Abs 490nm, Em 515nm, Extinction Coeficient 83,000 [cm.mole]-1.
Saturated Stock solution at pH 7 is around 200ug/ml (Jy9th97)

Dissolved a small amount of FMA in 300ul. Spin the solution to remove particular material. Measure the concentration by spec.

Incubate 5ul of packed RBC's in 100ug/ml in a final volume of 1ml, for 40 minutes. Washe 3x.


EMA Labeling Band 3

Eosin-5-maleimide (EMA) is a produte of Molecular Probes  E-118,  Absorbance 524nm,  Emission 541nm. E =100,000.   Molar extinction coefficient [cm.mole]-1. Molecular Weight 743g/mole
 

Incubate 10-30mL of washed/packed RBC's in EMA @80g/uL in a 1mL for 40 min. then wash in PBS/BSA 3x
 

Stock  Solution

Get EMA out of frezzer
Warm the bottle to room temp to avoid water condensing on the contents
Take out a tiny amount with clean stainless spactular
Dissolve in ~300ul of PBS into EMA
Dilute stock solution (as required) and measure concentration in the spec (see Spectrophotometer Protocol)

The volume of stock required to make 1ml @ 80ug/ml is:
(ViCi=VfCf)  Vi = 80[ug/mL] X 1000[uL] / EMA Stock [ug/ml]

Dilution

10x          100ul  of stock solution  add to 1mL
100x        10ul of stock solution add to 1mL


Spectrophometer Protocol

Place 200uL of PBS in each side for a baseline correction
Replace  R  side with EMA solution press mode #8 condition   (enter)
Mode  #2 spectum   (enter)
Parameter change   yes          600.0 & 450.0          (press start)
Data processing     yes
Press copy

Calculation

Conversion of concentration in absorbance to ug/ml.

Concentration [molar] = Asorbance [cm-1] * Dilutuion Factor/extinction coefficient [cm.mole]-1

Concentration [ug/ml] = Concentration [molar] * Mol.Wgt [g/mol] *1000


DIDS (Band 3)

4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, disodium salt (DIDS) Molecular Probes D-337, ( also from Aldrich, Sigma)
Molecular Weight: 498.47. Absorbance 350nm, Emission 460nm. Extinction Coefficient 36,000[cm.mole]-1.

DIDS binds covalently to Band3 and reportedly alters band3 tetrameric/dimeric equlibrium to favour the dimer which is less associated to ankyrin.

Washed RBC's at 50% hematocrit were incubated in DIDS @ 50-100µM at 37°C for   1 hours
DIDs has a high binding affinity. Binding saturates at 100µM within minutes.

100µMolar --> 5mg DIDS + 100ml H2O
Phil Low: DIDS has a 5µM binding constant and stops band 3 anion transport instantly via a non-covalent link. With extended incubation DIDS forms a covalent link with band 3. The rate of this covelent link increases with both pH and temperature. Phil suggests incubating in DIDS at 10µM at 37°C.


Rhodamine Phalloidin Labeling of Actin

Molecular Probes (R-415), Extinction Coefficient E=75,000 (cm.Molar)-1, methanol.

RhPh Preparation
*Measure the concentration of RhPh in methanol with the spectrophotometer. Ans: Absorbance / 75,000 (*106)=YcµMolar.
*Require: is to dry down the correct amount of RhPh in methanal so that when we rehydrate it with 20µl of (lysis) buffer the final concentration is 4-5µMolar.
Vi = Vf*Cf/Ci = 20µl*4µM/YµM = Yv ul
*Prepare many 1.5ml vials with Yvµl of RhPh in methanol and dry it down (in the dark). This can be stored at -20°C for weeks.

Lysis Buffer Preparation
*Red cells are osmotically lysed in a 7.5 mM phosphate (15-20 mOsm) PBS buffer which includes the RhPh and ATP (5.5 mg MgATP/10 ml). The ATP is required to maintain discotyal morphology after the cells are resealed.
*Dilute 1xPBS by 20 fold by volume with dH20.
* Add MgATP at 5.5mg/10ml (Sigma, A-9187)

Labeling Protocol
* Weigh out 0.0055g of MgATP, add 9.5 ml of dH2O, add 0.5ml (500ul) of 1xPBS. Check the osmolarity is 15-20mOsm. (Lysis Buffer with ATP)
* Rehydrate the RhPh with 20ul of lysis buffer
* On ice, Add 5ul of packed RBC's and incubate for 4minutes
* Reseal by adding 2.5ul of 10xPBS and incubate at 37°C for 30-60minutes
* Cell show show normal morphology and 20 hemat

To label actin, 4 mM rhodamine phalloidin (Molecular Probes, Eugene, OR) was prepared in 7.5 mM phosphate (20 mOsm) with 5.5 mg MgATP/10 ml. to a final volume of 20 ml. 5 ml of red cells were then added and the suspension incubated on ice for 4 min.  Afterward, 2.5 ml of 10x PBS was added, the cells incubated at 37°C for 30 minutes and then resuspended in 290 mOsm PBS/BSA.