Assessing the Assembly Competence of Purified Alpha- and Beta-Tubulin Isotypes
By: Harjot Kaur
University at Buffalo ’12
Mentors: Leah Miller & Eddie Nieves
Albert Einstein College of Medicine
Abstract
In this paper, we will describe how to purify chicken alpha and beta tubulin from
chicken erythrocytes. Then we will explain how we used the chicken extraction to
separate the alpha tubulin from beta tubulin and then again attempt to combine them
together. This method allows us to assign specific tubulin isotypes and post-translastional
modifications. This peptide has a mass of 2804.2, which is 107 Da higher than 1-Y. We
obtained a partial sequence of this peptide by MS/MS, but do not yet know the complete
sequence of this novel form. These results will be used to verify isotype masses of
chicken erythrocyte tubulin obtained from the developing method known as Limited
Cleavage.
Introduction
Microtubules are polymers that consist of - and tubulin subunits. Pervious
studies have shown that there are six -tubulin and seven- tubulin proteins present in
mammals, with most of the sequence diversity occurring at the C-terminus. These
different protein forms are called isotypes. Chicken erythrocyte microtubules in
particular, mostly consist of the 1 and VI tubulin isotypes. Furthermore, these tubulin
proteins can undergo post-translational modifications such as tyrosination/detyrosination
(+/-Y, +/- 163 Da), glutamylation (+E, +129 Da), and phosphorylation.
Microtubules are found in eukaryoticcells and are necessary for many cellular
processes. They are responsible for arranging the organelles in their correct location in
the cell, separating genetic materials, transferring material within the cell, and also
maintaining the shape of the cell. If microtubules are destroyed, it leads to the death of
the cell because without microtubules cells are unable to divide during mitosis.
Isoelectric focusing (IEF) combined with mass spectrometry is an established
method to identify tubulin isotypes. In this specific method, a chemical called cyanogen
bromide (CNBr) cuts the peptides at methionine, which releases the C-terminus of the
tubulin protein, also known as the isotype defining region. Based on the mass of this
peptide, tubulin isotypes and post-translational modifications can be assigned. Since
tubulin isotypes observed in this procedure are only identified by the C-terminal peptide
(~15-20 residues), any modifications or mutations occurring in other parts of the protein
would not be observed. Therefore, scientists are trying to create a new method in
identifying tubulin isotypes called Limited Cleavage, in which larger peptides will be
analyzed.
In IEF, protein bands are separated according to their isoelectric point (pI).
Isoelectric point is when the overall protein charge is zero. Protein charge is dependent
on the surrounding pH of the environment. When proteins are at pH values above their
pI, they are negatively charged and vice versa. During IEF, positively charged proteins
migrate towards the cathode whereas the negatively charged proteins move towards the
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anode until they reach their pI. Using a narrow pH gradient of 4.5-5.5 allows high
resolution of proteins with similar pI to be detected as distinct bands when IEF is
complete. Furthermore, tubulin proteins have pI within the range of 4.7 to 5.0.
Mass spectrometry measures the exact peptide masses. A mass spectrometer
consists of three main components: ion source, mass analyzer, and detector. The ion
source converts peptides into ions, the mass analyzer measures the mass-to-charge ratio
of the peptides, and finally the detector, detects the number of ions at each mass-tocharge ratio. There are many different ionization methods and mass analyzers. In this
paper the ionization source was matrix-assisted laser desorption ionization (MALDI) and
the mass analyzer was Time of Flight/Time of Flight (TOF/TOF). In our experiment, the
peptides were first converted into negative ions and then focused in a straight path using
a guide wire. Following which, the same amount of kinetic energy was applied to each
ion in order to measure the ion’s time of flight. This time of flight is used to determine
the masses; the longer the time the ion takes to reach the detector, the heavier the peptide
and vice versa.
Purpose
Separating α- and β-tubulin from microtubules and being able to reassemble them into the
same bead-like structure.
Methodology
Protocol for Tubulin Purification
Preparation of buffers
Citrate/saline: 3% sodium citrate/ 0.9% NaCl
1 Liter Prepared
Calculations
__ x__ = __3%_
1000mL
100%
x = 30g of Sodium Citrate
___x___= _0.9%_
1000mL
100%
x = 9g NaCl
Add enough dH2O until 900mL of solution
Mix well with a stirrer
Adjust pH to 7.41 with 5M HCl
Adjust solution level to 1000mL
Saline: 0.9% NaCl
1 Liter prepared
Calculations
_ _x___= _0.9%_
1000mL 100%
x = 9g NaCl
Mix well with a stirrer
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Adjust solution level to 1000mL
Phosphate/: 20mM Na2HPO4, 100mM L-glutamic acid
glutamate 500mL Prepared
Calculations
Na2HPO4 (Sodium phosphate dibasic, Sigmaultra minimum 99.0%)
MW=141.96g
141.96g  1000mL  1000mM
70.98g  500mL  1000mM
1.4196g  500mL 
20mM
C5H8NNaO4 x H2O (L-Glutamic Acid monosodium salt hydrate)
MW=169.1g
169.1g  1000mL  1000mM
84.55g  500mL  1000mM
8.455g  500mL  100mM
Add 1.4196g of Na2HPO4 and 8.455g of C5H8NNaO4 x H2O and mix
Adjust pH to 6.75
Add enough dH2O to reach 500mL of solution
EGTA: Molecular Formula = C14H24N2O10
50mL prepared
Calculations
MW = 380.4g
380.4g  1000mL  1000mM
38.04g  1000mL  100mM
1.902g 
50mL  100mM
Add 1.902g of EGTA to a 50mL beaker
Mix with a stirrer
Adjust pH to 7.5 with NaOH
Add enough dH2O to reach 50mL
MgCl2: 50mL prepared
Calculations
MW = 203.31g
203.31g 1000mL  1000mM
20.331g 1000mL  100mM
1.017g
50mL  100mM
Add 1.017g of MgCl2 to a 50mL beaker
Mix with a stirrer
Add enough dH2O to reach 50mL
GTP: 100mM solution prepared
Calculations
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MW=523.2g
Stock of .1g of GTP
How much H2O is needed to make 100mM of GTP?
523.2g  1000mL  1000mM
52.32g  1000mL  100mM
__.1g__= 1.912mL H2O for 100mM GTP
52.32g
100 x: 3.78mL of solution prepared
Nucleotides MW = 551.15 (ATP) x 0.24M = 132.2g/L
_0.5g_ = 3.78mL total solution
132.2g
0.5g ATP + 0.378mL of 0.1M GTP + 3.4mL H2O = 100x nucleotides
Transfer ~1mL of 100x nucleotides into 4 separate 1.5mL test-tube
DTT: 5mL prepared
MW=154.25g
154.25g  1000mL  1000mM
15.425g  1000mL  100mM
0.077g 
5mL  100mM
PIPES: C8H18N2O6S2
250mL prepared
MW = 302.4g
302.4g  1000mL  1000mM
75.6g  250mL  1000mM
37.8g  250mL  500mM
H1P: (Phosphate/ glutamate buffer with 0.1mM EGTA, 1mM MgCl2, 1mM
DTT, 0.1mM GTP, and 2.4mM ATP)
100ml phosphate/ glutamate added to a graduated cylinder
M1V1 = M2V2
EGTA
.1mM * 100mL = 100mMx
x = 100µL
MgCl2
1mM * 100mL = 100mMx
x = 1000µL
DTT
1mM * 100mL = 100mMx
x = 1000µL
GTP
.1mM * 100mL = 100mMx
x = 100µL
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Sucrose: (50mL phosphate/ glutamate with 43% sucrose, 1mM each EGTA, MgCl2,
Cushion DTT, and GTP)
50mL phosphate/ glutamate added to a graduated cylinder
EGTA and MgCl2
M1V1 = M2V2
1mM * 50mL = 100mMx
x = 500µL
500µL
Sucrose
__x__ = _43%_
50mL 100%
x = 21.5g of sucrose
DTT
Prepare 1000mM and 1mL
MW = 154.25g
154.25g  1000mL  1000mM
.15425g 
1mL  1000mM
1mM * 50mL = 1000mMx
x = 5µL
GTP
1mM * 50mL = 100mMx
x = 500µL
Explanation of Calculations
M1V1 = M2V2
M1 = molarity desired
V1 = Total volume desired
M2 = Molarity of stock
V2 = How much volume needed?
1. Blood is collected from eight chickens at 11am. A total volume of 800mL
including citrate/ saline solution of 300mL.
2. The blood is then filtered on a four layer cheese cloth for feathers and other
unnecessary solids in the blood.
3. The remaining filtered blood is now divided into two separate centrifuge
bottles(500mL)
4. Centrifuge used: Sorvall Lengend RT
At 3,500rpm
For 10min
At 22ºC
5. The supernatant is aspirated to remove plasma.
6. The remaining pellet is resuspended in 250mL saline solution each.
7. Centrifuge again with Sorvall Lengend RT at 3,500rpm for 10min at 22ºC.
8. Remove supernatant with aspiration.
9. Bottle#1
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Empty bottle = 147.306g
Bottle + blood = 225.606g
Weight of blood = 78.3g
Bottle#2
Empty bottle: 146.122g
Bottle + blood = 222.505g
Weight of blood = 76.38g
Total Weight of Blood = 154.68g
10. Wash the erythrocyte pellet with 0.6 volume of phosphate. Glutamate assembly
buffer.
Total mass = 154.68g
154.68 X 0.6 = 92.8mL of phosphate/ glutamate buffer
Total volume = 154.68g + 92.8mL ~ 250mL
Addition of EGTA and MgCl2
M1V1 = M2V2
1mM(250mL) = 100mMx
x = 2.5mL of EGTA and 2.5mL of MgCl2 added
Addition of GTP
0.1mM (250mL) = 100mMx
x = 250µL of GTP
17.5µL of 2-Mercaptoethanol added
11. The temperature is brought to 5ºC on an alcohol/ice bath.
12. Sonication is performed with a large probe.
- Sonicate on ice
- Use an 80mL beaker and place 40mL of sample each time
- Sonicate for 60sec. Rest for 30sec. Sonicate again for 45sec.
- Sonicate until color changes from red to black.
13. Separate 35mL of sample into 7 centrifuge tubes.
14. Centrifuge in Sorvall Centrifuge (rotor SS-34) at 4ºC for 60min at 15,000rpm to
remove nuclei and membrane debris.
15. 160mL of supernatant is collected and transferred to a clean flask.
16. Add 20% glycerol (40mL) and 2mL of 100x nucleotide.
17. Incubate for 45min at 37ºC.
18. Separate the erythrocytes into 6 centrifuge tubes (35mL each).
19. Centrifuge again using the same centrifuge (Sorvall rotor SS-34) for 90min, at
15,000rpm, at 30ºC.
20. 183mL of supernatant is obtained.
21. Pellet is frozen on dry ice at -80ºC.
22. Add 1/10 of phosphate/ glutamate buffer
183mL of supernatant.
183mL/10 = 18.3mL
6 centrifuge tubes
18.3mL/6 = 3.05mL of phosphate/ glutamate buffer in each sample.
23. Pellet is mixed with a glass/ Teflon tissue homogenizer.
24. Additional 2mL of phosphate/ glutamate buffer is added to each tube for washing.
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Total volume ~ 38mL
25. Incubate on ice for 30min to depolymerize microtubules.
26. Separate into 13 x 51mm polyallomer 12 centrifuge tubes
27. Use Beckman centrifuge Rotor TLA 120.3 at 62,000rpm for 45min at 4ºC.
Ran 6 tubes in two centrifuge each.
Remove supernatant into an empty flask.
Total supernatant = 33mL
28. Add 8.3mL of glycerol
Total volume = 33mL + 8.3mL = 42mL
Add GTP
0.1mM(42mL) = 100mMx
x = 42µL ~ 50µL GTP added
29. Place the sample onto a water bath at 37ºC for 45min.
30. Total volume = 42mL
Divide 10.5mL of sample into 4 centrifuge tubes for rotor 60 TI
31. 50mL of cushion (43% sucrose in phosphate/glutamate buffer) is already prepared
Add GTP to the cushion
0.1mM (50mL) = 100mMx
x = 50µL GTP
32. The supernatant after incubation was centrifuged on the 3 ml of cushion at
49.000rpm for 60min at 25˚C.
Mass of tube without cap
Tube 1 = 16.67g
Tube 2 = 16.89g
Tube 3 = 16.80g
Tube 4 = 16.93g
After centrifugation: Tube 1-16.79g
Tube2-17.00g
Tube3-16.92g
Tube4-17.06g
Total- Microtubule pellet is-200mg.
33. Microtubule pellet were resuspended in 6 ml 0.5M sodium PIPES assembly buffer
on ice, pooled in to two tubes and supplemented with 0.1 mm DDT-6µL; 10%
DMSO-0.6ml; 1mM EGTA-60µL. Mixture was incubated at 37˚C for 30 min. and
centrifuged at 45.000rpm for 35 min. at 25˚C (Rotor TI-60 Beckman).
Pellet were resuspended in 4 ml 0.1 m sodium PIPES ass buffer.
Incubate on ice 1 hr. Aliquot into 4 1.5ml tubes- 1ml in each. Tubes was frozen in
liquid nitrogen and stored at -80˚C.
34.OD was measure at 280 nm on Nanodrop.
Trial 1 = 4.93mg/mL
Trial 2 = 4.96mg/mL
Trail 3 = 4.92mg/mL
Average = 4.94mg/mL
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Results
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Discussion/Conclusion
Purification of chicken erythrocytes was a successful procedure. Over the course
of three days we obtained 200mg of tubulin pellet from the eight chickens. This amount
is highly considered an excellent extract from the chicken. However, we did face few
complications such as for the first procedure, the centrifuge did not work properly and we
had to perform the centrifugation serval times before we arrived with the desired results.
Using isoelectric focusing combined with mass spectrometry and cyanogen
bromide, we were able to determine the tubulin isotypes present in chicken erythrocytes.
Our results corroborated with the previous findings of Rudger and co-workers, who
identified 1-Y and VI as the major isotypes in turkey erythrocytes tubulin. However,
we found different modifications to these isotypes than described previously.
In each of the three gels 1-Y was the major alpha tubulin. A small amount of
1+Y was identified in gels 2 and 3. Rudger and co-workers also described a small
amount of alpha tubulin to be tyrosinated. Unlike, Rudger and co-workers, who did not
find tubulin modified by glutamlytion, we found evidence for both 1+Y and 1-Y with
one post-translationally added glutamate residue. This difference in the identification of
post-translational modifications may be due to higher resolution of the tubulin isotypes
on our 24cm of 4.5-5.5 IEF gel. This may also be due to new and more sensitive mass
spectrometry techniques that have been developed in the past 15 years, when initial
experiments were preformed. Rudger and co-workers used a different method in
obtaining and purifying tubulin peptides which may also account for some differences
seen in our and their experiment.
The only form of beta tubulin identified was the VI isotype and we did not
observe any post-translationally modified forms. The work done by Rudger, identified
approximately 10% of phosphorylation on Ser441 of bVI tubulin. Possible reasons for
not detecting phosphorylation might be that too little protein was loaded on the IEF gel.
Future experiments will provide a more rigorous analysis specifically focusing on the
detection of phosphorylation.
Unique to our experiment was a novel tubulin form that was detected at a pI
slightly lower than alpha 1-Y. In order to obtain sequence information for this peptide,
MS/MS data was acquired. Although the exact sequence is not yet determined the
MS/MS data indicates that it is related to alpha 1-Y and is 107Da higher in mass. This
could potentially be a new modification or isotype, which has not previously described
for tubulin.
While this is an established method, we needed to optimize the conditions for our
sample. In initial experiments, a smear emerged on the top and bottom of the Gel 1
where tubulin should have focused. Due to this contamination, we visualized a1-Y in
every band including beta tubulin bands. In order to obtain better results, we repeated the
experiment with Gel 2 containing the same amount of the protein as Gel 1 and Gel 3 was
loaded with 1uL of protein with the same concentration as Gel 1 and Gel 2. Better
resolution was achieved in both of these gels and the resulting data showed better mass
spectra. We also found de-salting samples on a ZipTip after cyanogen bromide cleavage
produced cleaner spectra. The mass spectra without ZipTip contained high amounts of
salts that were interfering in the visualizations, but after ZipTip, the spectra were clear.
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Using the information about tubulin isotypes obtained in this experiment will
allow us to readily predict the masses of the larger peptides resulting from Limited
Cleavage. IEF with mass spectrometry is a procedure well known to identify tubulin
isotypes and therefore, results from this particular chicken erythrocyte tubulin will be
used to verify the masses obtained from the chicken erythrocytes tubulin in Limited
Cleavage procedures.
Future Studies
Currently, we are re-doing the western blotting and the mass spec. due to the
contamination of alpha in the beta and vice versa.
We also hope to overcome the difficulties of reassembling the alpha and betatubulin into microtubules.
References
Miller, L. M., Menthena, A., Chatterjee, C., Verdier-Pinard, P., Novikoff, P. M., Horwitz,
S. B., and Angeletti, R. H. (2008) Increased Levels of a Unique Post-Translationally
Modified IVb-Tubulin Isotype in Liver Cancer. Biochem 47, 7572-7582.
Weber, K. and Rudiger, M. (1993) Characterization of the post-translational modification
in tubulin from the marginal band of avian erythrocytes. Eur. J. Biochem 218, 107-116.
Acknowledgements
I would like to thank Leah Miller and Berta Burd for being side by side while conducting
this experiment. I would also like to thank Eddie Nieves for allowing me to work under
his authorization. And also Dr. Ruth Angeletti, the director, as well as everyone in the
lab, for making my summer research experience, one to remember.