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Isolation and Purification of Acid Phosphatase

Isolation and Purification of Acid Phosphatase

Name of Student

Institution Affiliation

Isolation and Purification of Acid Phosphatase

Results

Table 1. Piece Protein Assay

Fraction Number Total Volume (mL) Dilution Used Average A595 Protein Conc. Dilute Fraction (mg/mL) *10^-3 Protein conc. Fraction (mg/mL)

*10^-3 Total Protein (mg)

*10^-5

1 0.05 1 Data error 1.2850 1.2850 6.425

2 0.05 1 Data error 1.3037 1.3037 6.5185

3 0.05 1 1153.0173 0.9451 0.9451 4.7255

4 0.05 1 294.7100 0.3567 0.3567 1.7835

5 0.05 1 316.9231 0.3804 0.3804 1.902

3 0.05 1 1102.5018 0.9290 0.9290 4.645

3 0.05 1 1136.7230 0.9401 0.9401 4.7005

4 0.05 1 318.4715 0.3820 0.3820 1.91

4 0.05 1 308.3141 0.3713 0.3713 1.8565

5 0.05 1 225.1985 0.2797 0.2797 1.3985

5 0.05 1 268.9936 0.3288 0.3288 1.644

1 0.05 2 Data error 1.08911 2.17822 10.8911

1 0.05 5 847.9720 0.8128 4.064 20.32

1 0.05 5 875.2789 0.8281 4.1405 20.7025

1 0.05 5 901.4716 0.8421 4.2105 21.0525

2 0.05 5 663.0801 0.6914 3.457 17.285

2 0.05 5 710.5997 0.7256 3.628 18.14

2 0.05

5 745.9015

0.7496 3.748 18.74

Table 2. Acid Phosphatase Activity

Fract

# Total volume

ml Dilution

used Volume

Used in assay(mL) Avg. A405 [PNP]

in

Assay

(M)*10^-2 Total

PNP

(mol)

*10^-2 [PNP]

Dilute Fraction

(mole/mL)

*10^-2 [PNP]

in

Fraction

(mol/mL) Units

mL Total

Units

1-1 0.05 40 0.1 0.3610 1.9202 7.68 1.896 0.7584 0.7584 3.07152

1-2

0.05

40

0.1

0.3505

1.8644

7.4576

1.8414

0.737

0.737

2.98485

2-1 0.05 40 0.1 0.3504 1.864 7.456 1.841 0.7364 0.7364 2.98242

2-2 0.05 40 0.1 0.3488 1.8553 7.4212 1.832 0.7328 0.7328 2.96784

3-1 0.05 40 0.1 0.3663 1.9484 7.7936 1.924 0.7696 0.7696 3.117

3-2 0.05 40 0.1 0.3669 1.9516 7.8064 1.9275 0.771 0.771 3.12255

4-1 0.05 40 0.1 0.3596 1.9128 7.6512 1.889 0.7556 0.7556 3.06018

4-2 0.05 40 0.1 0.3976 2.115 8.46 2.09 0.836 0.836 3.3858

5-1 0.05 40 0.1 0.3102 1.65 6.6 1.63 0.652 0.652 2.6406

5-2 0.05 40 0.1 0.2970 1.58 6.32 1.56 0.624 0.624 2.5272

9. Calculation of the concentration of p-nitrophenol(PNP) from the corrected average absorbance reading.

A = Elc where A- Absorbance

l- Path length

c- Concentration

E – Molar absorptivity

Therefore concentration, C = A/El

= 0.3610/(1.88 * 10^4) * 1

=1.9202 * 10^-5

C = A/El

= 0.3505/(1.88 * 10^4) * 1

= 1.8644 * 10^-5

Repeating this for all the values yields the concentration of all the other fractions.

10. The total number of moles of the PNP is a product of its concentration and the total volume of the assay mixture. The volume in this case includes the volume of potassium hydroxide which is 4.0 ml

Therefore, Total moles = Concentration of PNP * Total Volume of assay

For Fraction 1, Total moles = (1.9202 * 10^-5 ) * (4.0 * 1000)

= 7.68 * 10^-2 moles

For fraction 1-2, Total moles = (1.8644 * 10^-5) * (4.0 *1000)

= 7.4576 * 10^-2 moles

For fraction 2-1, Total moles = (1.864 * 10^-5) * (4.0 * 1000)

= 7.456 * 10^-2 moles

For fraction, 2-2, Total moles = (1.8553 * 10^-5) * 4000

= 7.4212 * 106-2moles

For Fraction, 3-1, Total moles = (1.9484 * 10^-5) * 4000

= 7.7936 * 10^-2 moles

For Fraction, 3-2, Total moles = (1.9516 * 10^-5) * 4000

7.8064 * 10^-2moles

For Fraction, 4-1, Total moles = (1.9128 * 10^-5) * 4000

= 7.6512 * 10^-2moles

For, 4-2, Total moles = (2.115 * 10^-5) * 4000

= 8.46 * 10^-2 moles

For, 5-1, Total moles = (1.65 * 10^-5) * 4000

=6.6 * 10^-2 moles

For 5-2, Total moles = (1.58 * 10^-5) * 4000

= 6.32 * 106-2 moles

11. moles PNP/ml diluted fraction = Total moles / Amount of diluted sample

= (7.68 * 10^-2)/4.05

= 1.896 * 10^-2

To get the volume of the undiluted solution, the moles PNP/mL of diluted fraction is multiplied by the dilution factor of 40

Therefore, moles of undiluted fraction = moles of diluted fraction * 40

= (1.896 * 10^-2) * 40

= 0.7584

12. One unit of enzyme activity produces 1mole of PNP in 15 minutes at 300c.

0.7584

1 unit of enzyme activity is equivalent to 1mole of PNP

Therefore 0.7584 mole is equivalent to 0.7584 units

This is repeated through all the twelve samples

The total number of units is the product of the units/mole multiplied by the total volume

0.7584 * 4.05 = 3.07152

Table 3. Purification of Acid Phosphatase

Fraction Total Volume (mL) Protein conc. (mg/mL) Total protein (mg) Total Activity (Units) Specific Activity (units/mg) % Recovery Purification (fold)

1 4.05 1.2850 6.425 3.07152 0.4781 47.81 1.4781

2 4.05 1.3037 6.5185 2.98242 0.458 45.8 0.458

3 4.05 0.9451 4.7255 3.117 0.66 66 0.66

4 4.05 0.3567 1.7835 3.06018 1.716 100 1

5 4.05 0.3804 1.902 2.6406 1.388 100 1

Discussion

The acid phosphatase has been successfully purified and isolated. The specific activity obtained ha at first shown significant change in the trend and the recovery has nearly attained a specific activity of nearly 47.81%. In table 3, it is clear that with each fraction addition, there was a significant decrease in the total protein that was to be produced. This significant decrease is due to increase in the amount of unwanted proteins in each step that follows. The use of p-nitrophenol was used to test for the enzyme in order to cleave the phosphate group. In addition to that, the use of Potassium hydroxide ensured the reaction turned yellow in the long run for the observable change to be clearly visible. With each subsequent procedure, the specific activity tended to increase. The increase indicates that the process was successful and it is similar to the expected trends (Vallee).

Dilution analysis was put into consideration at each step of the overall process. It was necessary to report the overall activity in the entire fraction. However, each step was not effective. It may be due to there being unintentional and excess dilution. Excess dilution would significantly lead to errors in enzyme activity. Enzyme purification is a solvent-sensitive process thus explaining the sources of the discrepancies. The results therefore indicate a successful experiment due to increase in enzyme activity from one step to the next. The 100% is not the acid phosphatase as it is expected. This may be due to the presence of solid pellets at the bottom of the equipment.

Reference

Vallee, B. L. (2012, December). Zinc biochemistry: a perspective. In Biotechnological Applications of Proteins and Enzymes: Papers Presented at a Conference Honoring the Sixtieth Birthday of Professor Ephraim Katchalski-Katzir, Held at Kiryat Anavim, Israel, May 23-27, 1976 (p. 223). Elsevier.

Islamic state

Islamic state

Name

Institution affiliation

Islamic State terrorism

Formally known as ISIL or else ISIS (Islamic State of Iraq and al Sham the Levant/Syria) or DAESH.This group is the largest group that has remain threat to united states as a country. It pedals north parts of Iraq and the Western parts of Syria, inside which it has molded its specific vicious regime that sanctions sharia rule. Founded by Abu Bakr al-Baghdadi, the Islamic Government has approved out performances of brutality so cruel that even Al Qaeda has convicted it. The IS group consents fighters from all over the nation, and inspires them to relocate to “al Sham” or paradise on world. The group has an apocalyptic image in which they have faith in that by apprehending convinced corporal territory in the section they will escort in glory. They inspire terrorist bouts within Western republics and will receive the adherence of anyone who curses to them. The group funds himself through the selling of items, lubricant, drugs, and slaves. They have indorsed themselves seriously through communal media via efficiently shaped videos of horrible actions like executions.

Bibliography

Al-Tamimi, A. (2015). The evolution in Islamic State administration: The documentary evidence. Perspectives on Terrorism, 9(4).

Islamic Movement of Uzbekistan (IMU)

Islamic Movement of Uzbekistan (IMU)

Name

Institution

Date

Islamic Movement of Uzbekistan (IMU)

The Islamic Movement of Uzbekistan is a group of militias from Uzbekistan and thus can clearly understand where their name came from. The group can be dated back to 1998 and has been an active cell group since then. The group had actually been in existence before 1998, but before then they were not really organized and consisted of a couple of different groups each having a different name. The group was formed to overthrow the government that was in power then as the government was said to be pro-communist and was headed by President Karimov (Abdullaev, 2011). The group was formed by Thir Yuldashev who an Islamic Ideologue in collaboration with Juma Namangani was who was a former Soviet trooper. They felt it was time to overthrow the government and establish an Islamic state governed by sharia laws.

In 1999, the group conducted several attacks in Tashkent which was the capital of Uzbekistan as a way to destabilize the government and assassinate president Karimov. Karimov responded by becoming tougher on Muslim individuals especially to the conservant Muslims that stayed in Fergana valley. This, however, had adverse effects. More people joined the IMU and became stronger. From then they were able to conduct successful attacks. These raids impacted central Asia, and there was international pressure to expel IMU from their already established base. Russians flew Namangani and his soldiers to Afghanistan, Karimov saw this as a betrayal as he though the Russians were helping IMU. The group created a close affiliation with Taliban and Al-Qaida which are also notorious terrorist groups known for carrying out attacks especially in the US (Zen, 2016). While in Afghanistan and with permission from Taliban to operate, they set up their offices and training camps and started recruiting people. By 200, IMU had the number of people, equipment ranging from sniper rifles to night-vision goggles. Transport had also been improved as bin-laden ad provided them with helicopters.

With the right amount of people and fighters, Namangani conducted an attack in Batken in Kyrgyzstan and some northern parts of Tashkent. They also kidnaped 4 US mountain climbers and held them hostage in exchange for ransom from the US government. This angered the US government and was declared as a foreign terrorist organization. Ties between IMU and Taliban became even stronger that Namangani was said to be the deputy defense minister of Taliban Government. They even provided soldiers to Taliban during the campaign on Masoud.

After the 911 attacks in the US that are considered the deadliest attacks, the US government decided to invade Afghanistan and try to quash the terrorist cells especially Al-Qaida and Taliban. The IMU training camps were largely destroyed and most of their soldiers killed. Namangani, their leader was also killed during this period by a US airstrike. They retreated to tribal areas of Pakistan and laid low for a while. They, however, continued recruitment to try and build the organization up again. The Uzbek ethnic religion was mostly the members if the group but when their recruitment base expanded to Afghanistan, Tajikistan and other parts of Central Asia other ethnic groups joined (Carlisle, 1995). Their leaders now even came from other groups such as Kyrgyz who was a military commander in Abbas Mansur, Abu Zar al-Burmi who was a Pakistani. Other commanders included Usman Ghazi, Abu Usman Adil, and Tahir Yuldashev. In 2010, Abu Usman Adil became the new leader following the death of Tahir Yuldashev. Adil was however killed in 2012 in a drone strike by the US military. He was replaced by his deputy Usman Ganzi who was killed in 2015, their current leader is not yet known.

There are several systems that were used by the Arabs and Muslims in recruitment of youth to join IMU. They used to offer dirhams to individuals who attended Friday prayers in the mosque. It is in the mosque that they taught radical ideas. These radical ideas included telling the youth of how they will be rewarded with nine virgins if they killed non-Muslims or participated in the holy war. They also kept blaming the US government for their troubles such as the death of children and women due to the airstrikes. These preaching were enough to stir hatred among the youths, and most were ready to dedicate their lives to the holy war. The mosque is the most notorious location where the recruitment of youths take place. They use the religion to justify their acts, and many fall victims to this. They believe that if they die fighting this war, they will be heavily rewarded. Their media has also been another form of recruitment through the spread of the propaganda. It is known as Jundallah Studio and aside from airing their content they produced audio, newsletters to help draw more people to the group (Moore, 2007).

Just like all other terrorist groups, IMU needs to money to carry out their day to day operations. Their common source is the money paid by residents in the territories they control which can be considered as the tax we pay our governments. They also engage in criminal activities such as smuggling of drugs and selling them to get money (Wayback, 2012). They have also obtained funding from well-established groups such as Al-Qaida and Taliban. Irfan Demirtas was arrested due to the links that he was aiding the IMU in raising funds through fundraising. He was an Irish/US citizen who used his western influence to source money for the group

There are several attacks that IMU have taken acknowledgment of (The Investigative Project on Terrorism, 2010). In 2004, they were accused of the Tashkent bombing leaving 47 people dead and a dozen injured. In 2006, they attacked supporters of Tajik’s President Imomali. In 2011, they were blamed for the attack in Toloqan. In 2012, they took responsibility for the bombing of Peshawar Airport, 2014; they attacked Jinnah International Airport in Pakistan killing 39 people. They have not been involved in most international attacks although it is said that they helped Al-Qaida conduct the 9/11 attack on the US soil. They have played a great role in attacks in Pakistan, Uzbekistan, and Afghanistan.

In a nutshell, Islamic Movement of Uzbekistan played a major role in attacks especially locally and just helped groups like al-Qaida to conduct international attacks. They were able to run the organization smoothly and recruit quite some individuals into the group. With the right amount of personnel and armor, they were a feared group for quite a while. The US invasion of Afghanistan may have marked its slow extinction and eventually it’s extinction. With the death of their leaders and the leadership wrangles, the group became divided, and the attack from Taliban was almost thee final blow. The group may be regrouping again under new leadership and is trying to take control of various regions in Uzbekistan although with the war by US and Uzbekistan government against them it may be hard.

BIBLIOGRAPHY Abdullaev, E. (2011, July 16). Islam Sng. Retrieved from Islam Sng: http://www.islamsng.com/uzb/dialogue/2101

Carlisle, D. (1995). Geopolitics and Ethnic Problrs of Uzbekistan and it’s Neighbouts. In Muslim Eurasia: Conflicting Legacies, 71-103.

Moore, C. (2007). Combating Terrorism in Russia and Uzbekistan. Cambridge Review of International Affairs, 1-19.

The Investigative Project on Terrorism. (2010, March 4). The Investigative Project on Terrorism. Retrieved from The Investigative Project on Terrorism: https://www.investigativeproject.org/profile/133/islamic-movement-of-uzbekistan-imu

Wayback, M. (2012, June 18). Eurasia Critic. Retrieved from Eurasia Critic: http://www.eurasiacritic.com/articles/drug-trafficking-uzbekistan

Zenn, J. (2016). The IMU is extinct: what next for Central Asia’s jihadis? The Central Asia-Caucasus Analyst.