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Laboratory Class 4: Lipids and membranes 2024 70 PROCEDURES In experiment 1 you will prepare two membrane fractions from calf liver tissue: an endoplasmic reticulum (ER) enriched fraction (microsomal fraction) and a mitochondrial enriched fraction. You will extract the lipids from the microsomal fraction and separate them by TLC. A sample of microsomal lipids will be provided so that you can start the TLC separation early in the lab session. In experiment 2 you will analyze the lipid/ protein ratio of the mitochondrial fraction. Before performing these experiments, you should watch the following videos: Overview Homogenization (0:51) or (http://www.youtube.com/watch?v=NsYRQg40ZSQ) Homogenization (3:34) or (http://www.youtube.com/watch?v=mZnLdYSV830) Centrifugation (3:57) or (http://www.youtube.com/watch?v=5wCBHlAeoNw) Rotovapour (2:42) or (http://www.youtube.com/watch?v=PPmITKfBwOo) TLC (14:06) or (http://www.youtube.com/watch?v=IbU5l0DZD_Q) You should also perform this simulation: TLC: effect of solvent polarity (https://elearning.cpp.edu/learning-objects/organic-chemistry/tlc/?page=simulation.html) Finally, you should watch these animations: Centrifugation
2D-TLC
The animations are located in the Laboratory 4: Lipids and membranes
folder of the Laboratory sessions
module on the Brightspace site of the Laboratory component.
Laboratory Class 4: Lipids and membranes 2024 71 EXPERIMENT #1: Lipids from the microsomal fraction of the liver a) Lipid extraction 1.
Mince 2g of calf liver tissue with scissors and place it in 10 mL of ice-cold homogenization buffer (10mM Tris, 0.25M sucrose, 1mM EDTA, 1 mM DTT, pH 7.5). 2.
Homogenize the tissue on ice with a glass-teflon homogenizer. Pass the pestle up and down about 10 times. (Consult your TA). 3.
Transfer the homogenate to a 15 mL Corex tube and centrifuge at 1,000xg for 10 min at 4°C. CAUTION!
Tubes should be paired, and their weight balanced with homogenization buffer. 4.
Filter supernatant 1 through mira-cloth to remove lipid granules. Collect the filtrate in a 25 mL cylinder, measure its volume and transfer it into a new 15 mL Corex tube. Discard pellet 1 which consists mostly of tissue debris, unbroken cells and nuclei (
Figure 2
). 5.
Centrifuge supernatant 1 in the Corex tube at 25,000xg for 10 min at 4°C. CAUTION!
Tubes should be paired and balanced. 6.
Transfer supernatant 2 into a 15 mL Falcon tube. This supernatant contains the microsomes (
Figure 2
)
.
Set aside pellet 2 for lipid and protein assay in experiment 2 (steps 22-27). 7.
Transfer 3.2 mL of supernatant 2 from step 6 into a 50 mL Falcon tube and add 12mL of CHCl
3
:MeOH (1:2 v/v). Mix well using a Vortex. (In the fume-hood!)
. 8.
Centrifuge at 3000xg for 5min in a Sorvall Legend centrifuge (swinging bucket). (
Tubes should be paired!
). CAUTION!
Chloroform and methanol are toxic, especially CHCl
3
; they may irritate skin, eyes and respiratory tract. Use gloves and safety glasses. Avoid inhalation. (
http://www.sciencelab.com/msds.php?msdsId=9927133 Methanol MSDS
)
Laboratory Class 4: Lipids and membranes 2024 72 9.
Transfer supernatant to a new 50 mL Falcon tube. (Discard the pellet). Add 4 mL of CHCl
3
and 4 mL of water. Mix well using the Vortex. (In the fume-hood!)
. CAUTION! All handling of chloroform in this lab should be done, when possible, in the fume-hood, using proper gloves. 10.
Centrifuge at 3000xg for 5 min using the Sorvall Legend centrifuge (swinging bucket). (
Tubes should be paired!
) 11.
Using a transfer pipette, transfer the lower phase into a 100 mL round bottom flask. (Do not take any liquid from the top phase). 12.
Evaporate the chloroform in a rotary evaporator. (Consult your TA). Dissolve the residue in 0.5 mL CHCl
3
and transfer to a 1.5 mL microtube. Label the tube with the date, your lab section and group number, and hand it to your TA. Total lipids can be extracted in a similar way using the liver homogenate from step 2. Alternatively you can perform the tissue homogenization in CHCl
3
:MeOH (1:2 v/v). By adding 1/3 volume of water and 1/3 volume of CHCl
3
the mixture separates in two phases. As before, the bottom phase (CHCl
3
) contains the lipids. b) Two-dimensional TLC 13.
Each student
will perform a 2D-TLC separation of a microsomal lipids sample. The sample will be provided at the beginning of the lab session so that you can start the TLC separation right away and finish it within the lab period. 14.
Label an activated (heated at 100°C for 1-2 hours) 20 x 20 cm Silica G plate using a TLC template and a pencil. Mark only the spots, lines and traveling direction of the two developing solvents as seen in Figure 3
. On the top left corner label your plate with the date, your sections and group numbers.
Laboratory Class 4: Lipids and membranes
2024
73
15. Pipet 5 L of the standards and 10 L of the sample at the positions indicated in Figure 3
. Keep the spots as small as possible to increase resolution. Lipid standards:
1) CL (cardiolipin) and CH (cholesterol)
2) FA (fatty acid) and PC (phosphatidylcholine)
3) PI (phosphatidylinositol) and PE (phosphatidylethanolamine)
Which standard in each couple will run furthest?
Figure 3. TLC Template. Schematic depicting the labeling and loading of the 2D-TLC plate for experiment 1. 3
2
1
3
2
1
standards
2.5 cm
sample
standards
5 cm
5 cm
2.5 cm
ID
2
nd
dimens.
1
st
dimens
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Related Questions
Course Code: BIO121
Course Name: Principles of Biology I
Short Question:-1
(a)
Cells that secrete hormones and enzymes (e.g., pancreatic cells, stomach cells) have a lot of rough endoplasmic
reticulum. Explain why.
(b)
(i)
List THREE (3) cytoskeletal elements.
(ii)
Describe TWO (2) functions of cytoskeletons.
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Transport Across the Cell Membrane Worksheet
1. For each of the following examples, state whether the solution is isotonic, hypertonic or
hypotonic and draw an arrow to indicate which way water will move.
a)
b)
100% H₂O
10% NaCl
0% NaCl
90% H₂O
83
5% salt
10% salt
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cell defense worksheet (1) [Compatibility Mode]
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Step 13: Take the "Energy and Transport Challenge!"
How many ATP did you use?
What type(s) of protein(s) were used?
Explain when each type was used.
Step 14: Take the "Osmosis Challenge!"
What is Osmosis?
What is the name of the special proieins that let water pass through?
Is this passive or active transport?
Step 15: From your Scores Sheet record:
Lab Score (% correct):
Number Correct:
Numbar Incen et
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CELL STRUCTURE AND FUNCTION – EUKARYOTES, PROKARYOTES AND VIRUSES-LABORATORY REPORT SHEET
ACTIVITY 2: CELL MEMBRANES, OSMOSIS AND DIFFUSION
Initial Volume (Vi)
(mL)
Final Volume (Vf)
(mL)
Change in Volume
(Vi – Vf)
(mL)
A
20% sucrose
20% sucrose
92.0 mL
94.0 mL
-2.0 mL
B
40% sucrose
20% sucrose
92.0 mL
92.0 mL
0.0 mL
C
20% sucrose
40% sucrose
92.0 mL
90.0 mL
2.0 mL
Treatment
Percent Change
A
-2%
B
0%
C
2%
Hypothesize which dialysis bags should gain, lose or stay the same in regards to volume. Explain.
Which solution is hypotonic, hypertonic and isotonic?
Explain what the change in volume of the dialysis tube indicated and describe what happened when the volume increased and when the volume decreased.
Explain the relationship between solute concentration and amount of free water or water available for movement.
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32
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76
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Activity 3b - Diffusion across a semipermeable membrane
1. Define selective permeability.
2. Explain the properties of the cell membrane that make it selectively permeable.
Activity 3c - Osmosis in potatoes
1. Define osmosis.
2. Define hypertonic, hypotonic, and isotonic.
Hypertonic
Hypotonic
Isotonic
Activity 3d - Osmosis in plant cells
1. Fill in the table below to indicate what happened to the elodea cells in each of the following
solutions.
Solution
Hypertonic
Isotonic
Hypotonic
Will the cell membrane
swell or shrivel?
What happens to the chloroplasts in the hypotonic
solution? Why do you think that happens?
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question:
Increasing the rate of breathing is a more effective strategy to increase alveolar ventilation than increasing depth of breathing.
answer should clearly state whether or not the statement is correct and then concisely explain why. the answer should be like a few sentences and address all of the points in the statement.
Here is an example:
Both transmembrane carrier proteins and transmembrane channel proteins can mediate active transport of a hydrophilic solute through a cell plasma membrane.
This statement is incorrect. Movement of a solute through a channel protein is always passive, whereas carrier-mediated transmembrane transport can be either passive or active. A transmembrane channel protein creates a pore through the membrane allowing for simple diffusion of a hydrophilic solute down a concentration gradient through the membrane. In contrast, transmembrane carrier protein interacts with and 'escorts' a hydrophilic solute through the membrane and is capable of…
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Exercise 3
The MTT assay is a widely used assay to study cell viability. It measures cellular metabolic
activity, which serves an indicator of cell viability, proliferation and cytotoxicity. This non-
radioactive colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals
by metabolically active cells. Viable cells contain NAD(P)H-dependent oxidoreductase
enzymes that reduce the MTT to formazan. The insoluble formazan crystals are dissolved and
the resulting coloured solution is quantified by measuring absorbance at 500-600nm using a
multi-well spectrophotometer. The darker the solution, the greater the number of viable,
metabolically active cells!
Exercise 3.1 MTT protocol
Your colleagues in the School of Biology and Environmental Science discovered an unusual
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9:54 AM
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Prelab 7 - Tagged (2 of 64)
三:
Prelab 7 - Protein Catabolism
In this lab we focus on another important group of metabolism processes: the breakdown of proteins and amino acids. In
these labs we will utilize a series of media in tubes that differ in the protein for the bacteria to breakdown, and in the end
products they can detect. There are a few new vocabulary terms. Proteolytic enzymes are enzymes that breakdown proteins,
usually by first breaking apart the polypeptide into individual amino acids. Hydrolysis reaction is used to break apart the
peptide bond holding the amino acids in the polypeptide together. Hydrolysis is used to breakdown urea, a compound
produced by the body from the destruction of proteins.
Once broken down to individual amino acid, often the amine (NH2) functional group is broken off one end in a deamination
reaction, creating NH3. In a decarboxylation reaction the carboxyl group is removed, creating CO2. In a desulfuration
reaction a sulfur is…
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Question 6
Which ion is actively transported through the cell membrane and why?
O Na, because the ion is being moved against its gradient
O Mg, because the membrane is impermeable to this ion
OK, because the ion is being moved with its gradient
O Cl, because the ion is moved to equilibrium
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The diagram below shows three different proteins embedded in a cell membrane, as well as the concentration gradient surrounding the cell.
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Which of the following statements correctly states the process shown in the diagram?
O The process labeled 1 shows how cells can easily move molecules with the concentration gradient using high energy molecules.
The process labeled 2 shows how cells move molecules against the concentration gradient without needing high…
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Osmosis Pre-lab Question
Why don’t red blood cells swell or shrink in blood?
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Exercise 3
The fate of the synthesized proteins
A protein A is detected in a cell in various cellular structures among which the nucleus, the results
appear in the following table:
Time
5 min
10 min
20 min
40 min
Organelte
Nucleus
RER
Golgi A.
Vesicles
(+) = presence
(-) = absence
1. Does protein A undergo exocytosis? Justify based on the table.
2. According to your acquired knowledge, and with the help of the table above, indicate the fate
of the synthesized proteins.
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I'm waiting.... thank you
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a cell defense worksheet (1) [Compatibility Mode]
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What does polar mean? Hydrophilic
Step 8: Insert channel proteins into the membrane. Transport substances across the
membrane. Note: You can only transport substances using channel proteins until there were
What is this process called?
Step 9: Moving from
to
concentration requires the use of energy to
substances. This is called
transport and uses: (place answer in table)
1.
2.
Which is cell
Step 10: Carbohydrates are like identification badges. Cells that have different membrane
The immune system uses the carbohydrates to
carbohydrates do different
that your cells belong to
and are not
or other foreign cells.
What does the immune system do to foreign invaders?
149%
E Focus
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505 words
English (United States)
MAR
15
MacBook Air…
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this question from bioengineering
asap
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Lecture 6-Worksheet
Part 1: Below are TWO ER bound proteins. The green region is indicating the ER transfer signals.
1. Can you label the start and stop transfer signals based on the rules we just went over?
2. Draw and label these proteins.
(1)
(2)
N
N
ER lumen
Part 2: Sketch the orientation of this protein as it would appear in the transport vesicle and plasma
membrane.
membrane-enclosed
organelle
(e.g., ER or Golgi)
vesicle
plasma
membrane
1₂:0.
0-9
?
CYTOSOL
extracellular fluid
165
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answer only question g
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Tube
#
2c
Table 3-Determination of the optimum temperature of catechol oxidase enzyme.
1st Absorbance
0 min. at Expt.
Temp.
3c
4c
Q12) After 10 min at experimental temperature was enzyme activity high or low at 75°C and 100°C?
Support your answer by looking at the difference between 2nd and 1st absorbances. (
5c
Q13) Was the enzyme denatured at 75°C and/or 100°C% Support your answer by looking at what
happens after returning enzyme to room temperature AFTER enzyme was subjected to experimental
temperatures of 20 °C,40 °C,60 °C,80 °C and 100°C. "¹
/1
Q14) What happens to the enzyme structure and function when it becomes denatured? (
Potato Expt.
extract Temp.
(mL)
2
2
2
2
room
temp
20 °C
40 °C
75 °C
Boiling
100 °C
dH₂O Catechol
(mL) (mL)
0
0
0
0
13
||
13
13
13
Start Time: 4:20
Absorbance:
0.072
Start Time: 4:25
Absorbance:
0.114
Start Time: 4:25
Absorbance:
0.132
Start Time: 4:27
Absorbance:
0.097
2nd Absorbance
after 10 min. in Expt
Temp.
Time for reading: 4:30
Absorbance:
0.128
Time…
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Example Problem: FRAP Data Interpretation
The diffusion rate of four different membrane proteins (A, B, C, and D) was measured using a FRAP
experiment with purified liposomes. The FRAP recovery curves are shown below.
Fluorescence
intensity
ROI
A
3
Time
B
Time
Post-bleaching
imaging
с
Time
D
Time
Pre-bleaching Bleaching
imaging
imaging
(a) Which membrane protein exhibits the higher rate of diffusion in the lipid bilayer? A or B? C or D?
(b) Explain the most likely cause of the difference in the recovery curves for proteins A and C.
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Cell growth kinetics
A mouse-mouse hybridoma cell line is used to produce monoclonal antibody. Growth in batch culture is
monitored with the following results.
Time (days)
Cell concentration (cells.ml* x 10°)
0.0
0.45
00.2
0.52
0.5
0.65
1.0
0.81
1.5
1.22
2.0
1.77
2.5
2.13
3.0
3.55
3.5
4.02
4.0
3.77
4.5
2.20
(a) Determine the specific growth rate during the growth phase.
(b) What is the culture doubling time?
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Solve the whole question in the photo
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2
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I need help with the diagrams for aHypertonic, Isotonic, Hypotonic, Solute
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Course Code: BIO121
Course Name: Principles of Biology I
Read the question carefully first
Answer the question-4 chemical reaction according to the question 4(a) and 4(b) with step by step
clear explanation.
Question 4
(a)
In cellular respiration, glucose is broken down and the energy released is used to make ATP molecules.
ATP molecules can be produced via this reaction: ADP+ P; → ATP.
Is the production of ATP endergonic or exergonic?
(b)
What is the function of ATP molecules in cells?
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Velithin a ceu to the exterior 6s' the ceu
4) A cell from a human heart is placed in a solution containing 90% water. The
cell itself has a solute concentration of 30%.
a) Sketch the shape of the cell before and after it is placed in the solution for
a prolonged period of time. ( /2TI)
AFTER
BEFORE
b) What type of environment was the cell placed in?
( 1TI)
c) Which word can describe what took place in this cell?
( /1TI)
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Ethanol or
Lactic acid
G3P
Руruvat
Glucose
G3P
Руruvat
Acetyl-CoA
Citric Acid
Охaloacetate
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Question 8
What is true of binding pockets?
They contain electrostatic interactions
O They occur in unfolded proteins
O They only occur in the membrane
They contain variable amino acids
All of the above
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BSC1010C
Enzymes & Cellular Regulation
Dr. Harris
4. Compare the rate of the pepsin-catalyzed reaction at pH = 1.5 with the rate of the lipase-
catalyzed reaction at pH = 1.5.
5. Compare the rate of the pepsin-catalyzed reaction at pH = 8.0 with the rate of the lipase-
catalyzed reaction at pH = 8.0.
6. Based on your understanding of protein structure, explain in detail the effect of exposing an
enzyme to a pH outside its optimal range. Include a discussion of the effect on both the
structure and function of the enzyme.
7. At what pH values is lipase likely to be denatured? Explain your answer.
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4 (d-g)
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9:56
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PRE-LAB QUESTIONS
Sell anything Start building
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1. Compare and contrast diffusion and osmosis.
2. Draw a picture of a cell in isotonic, hypotonic, and hypertonic states.
3. Why don't red blood cells swell or shrink in blood?
yith
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Exercise 9 Q10(2)
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- CELL STRUCTURE AND FUNCTION – EUKARYOTES, PROKARYOTES AND VIRUSES-LABORATORY REPORT SHEET ACTIVITY 2: CELL MEMBRANES, OSMOSIS AND DIFFUSION Initial Volume (Vi) (mL) Final Volume (Vf) (mL) Change in Volume (Vi – Vf) (mL) A 20% sucrose 20% sucrose 92.0 mL 94.0 mL -2.0 mL B 40% sucrose 20% sucrose 92.0 mL 92.0 mL 0.0 mL C 20% sucrose 40% sucrose 92.0 mL 90.0 mL 2.0 mL Treatment Percent Change A -2% B 0% C 2% Hypothesize which dialysis bags should gain, lose or stay the same in regards to volume. Explain. Which solution is hypotonic, hypertonic and isotonic? Explain what the change in volume of the dialysis tube indicated and describe what happened when the volume increased and when the volume decreased. Explain the relationship between solute concentration and amount of free water or water available for movement.arrow_forward32arrow_forward76arrow_forward
- Activity 3b - Diffusion across a semipermeable membrane 1. Define selective permeability. 2. Explain the properties of the cell membrane that make it selectively permeable. Activity 3c - Osmosis in potatoes 1. Define osmosis. 2. Define hypertonic, hypotonic, and isotonic. Hypertonic Hypotonic Isotonic Activity 3d - Osmosis in plant cells 1. Fill in the table below to indicate what happened to the elodea cells in each of the following solutions. Solution Hypertonic Isotonic Hypotonic Will the cell membrane swell or shrivel? What happens to the chloroplasts in the hypotonic solution? Why do you think that happens?arrow_forwardquestion: Increasing the rate of breathing is a more effective strategy to increase alveolar ventilation than increasing depth of breathing. answer should clearly state whether or not the statement is correct and then concisely explain why. the answer should be like a few sentences and address all of the points in the statement. Here is an example: Both transmembrane carrier proteins and transmembrane channel proteins can mediate active transport of a hydrophilic solute through a cell plasma membrane. This statement is incorrect. Movement of a solute through a channel protein is always passive, whereas carrier-mediated transmembrane transport can be either passive or active. A transmembrane channel protein creates a pore through the membrane allowing for simple diffusion of a hydrophilic solute down a concentration gradient through the membrane. In contrast, transmembrane carrier protein interacts with and 'escorts' a hydrophilic solute through the membrane and is capable of…arrow_forwardExercise 3 The MTT assay is a widely used assay to study cell viability. It measures cellular metabolic activity, which serves an indicator of cell viability, proliferation and cytotoxicity. This non- radioactive colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. Viable cells contain NAD(P)H-dependent oxidoreductase enzymes that reduce the MTT to formazan. The insoluble formazan crystals are dissolved and the resulting coloured solution is quantified by measuring absorbance at 500-600nm using a multi-well spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells! Exercise 3.1 MTT protocol Your colleagues in the School of Biology and Environmental Science discovered an unusual looking green algae on a recent fieldtrip to Costa Rica. They have provided you with some extracts from the plant…arrow_forward
- 9:54 AM Done Prelab 7 - Tagged (2 of 64) 三: Prelab 7 - Protein Catabolism In this lab we focus on another important group of metabolism processes: the breakdown of proteins and amino acids. In these labs we will utilize a series of media in tubes that differ in the protein for the bacteria to breakdown, and in the end products they can detect. There are a few new vocabulary terms. Proteolytic enzymes are enzymes that breakdown proteins, usually by first breaking apart the polypeptide into individual amino acids. Hydrolysis reaction is used to break apart the peptide bond holding the amino acids in the polypeptide together. Hydrolysis is used to breakdown urea, a compound produced by the body from the destruction of proteins. Once broken down to individual amino acid, often the amine (NH2) functional group is broken off one end in a deamination reaction, creating NH3. In a decarboxylation reaction the carboxyl group is removed, creating CO2. In a desulfuration reaction a sulfur is…arrow_forwardt be submitted on one attempt auto-submit when time expires bondus password is 515b73158335f6 Question 6 Which ion is actively transported through the cell membrane and why? O Na, because the ion is being moved against its gradient O Mg, because the membrane is impermeable to this ion OK, because the ion is being moved with its gradient O Cl, because the ion is moved to equilibrium < Previousarrow_forwardHBOCPS Das X + Orders - X ← → C D Login OCPS Performance Matters Question 5 of 16 Question 1-5 lipid bilayer low concentration Performa X < Previous olaocps.performancematters.com/ola/ola.jsp?clientcode=flocps# Join a Game - Quizizz K! Play Kahoot! - Enter... Dashboard Submit Test 1 channel protein Educator 3 2 x 12 Exam: 01 × Plans for x carrier protein 3 The diagram below shows three different proteins embedded in a cell membrane, as well as the concentration gradient surrounding the cell. high concentration POREI ***** energy Performance Matte... AP EL U.S. Citize X FLVS Login My Quizzes - Quizizz Welcome, Asheley! 02.Cl.Biology.CRM3.2_2023 EL https://sex S4JypvF Which of the following statements correctly states the process shown in the diagram? O The process labeled 1 shows how cells can easily move molecules with the concentration gradient using high energy molecules. The process labeled 2 shows how cells move molecules against the concentration gradient without needing high…arrow_forward
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