1 What would be the molar concentration of the following compounds needed to prepare a solution
which is isotonic with physiological saline?
(a) KCl
(b) CaCl2
(c) glucose
(d) sucrose
2 Answer the following questions related to making
Buffer A is 100 mL of solution containing 5 mL 1 M Na2HPO4 and 5 mL 1 M NaH2PO4. pH 6.82.
Phosphate has a pKa of 6.82.
Addition mmol added ratio [base]:[acid] pH
4 mL HCl 4 1 : 9 5.87
3 mL HCl 3 2 : 8 6.22
2 mL HCl 2 3 : 7 6.45
1 mL HCl 1 4 : 6 6.64
no additions 0 5 : 5 6.82
1 mL NaOH 1 6 : 4 6.99
2 mL NaOH 2 7 : 3 7.19
3 mL NaOH 3 8 : 2 7.42
4 mL NaOH 4 9 : 1 7.77
Buffer B is 100 mL of solution containing 5 mL 0.5 M Na2HPO4 and 5 mL 0.5 M NaH2PO4.
pH 6.82. Phosphate has a pKa of 6.82.
Addition mmol added [base] : [acid] pH
4 mL HCl 4 1.5 mmol unbuffered H+ 1.82
3 mL HCl 3 0.5 mmol unbuffered H+ 2.33
2 mL HCl 2 0.5 : 4.5 5.87
1 mL HCl 1 1.5 : 3.5 6.45
no additions 0 2.5 : 2.5 6.82
1 mL NaOH 1 3.5 : 1.5 7.19
2 mL NaOH 2 4.5 : 0.5 7.77
3 mL NaOH 3 0.5 mmol unbuffered OH- 11.67
4 mL NaOH 4 1.5 mmol unbuffered OH- 12.18
Buffer C is 100 mL of solution containing 10 mM Tris. pH 6.82. Tris has a pKa of 8.08
Addition mmol added [base] : [acid] pH
4 ml HCl 4 3.5 mmol unbuffered H+ 1.46
3 ml HCl 3 2.5 mmol unbuffered H+ 1.6
2 ml HCl 2 1.5 mmol unbuffered H+ 1.82
1 ml HCl 1 0.5 mmol unbuffered H+ 2.33
no additions 0 0.5 : 9.5 6.82
1 ml NaOH 1 1.5 : 8.5 7.33
2 ml NaOH 2 2.5 : 7.5 7.6
3 ml NaOH 3 3.5 : 6.5 7.73
4 ml NaOH 4 4.5 : 5.5 7.99
5 Shown below are some Western Blots in a system designed to detect a protein X (130 kDa) in cell
extracts. The primary antibodies used were rabbit poly-clonal anti-X and mouse monoclonal antiGAPDH (GAPDH (glyceraldehyde-3-phosphate dehydrogenase, (38 kDa) is a housekeeping gene
whose levels are not expected to change in a particular tissue type under different treatments). The
secondary antibodies used were goat polyclonal anti-rabbit Ig Peroxidase-conjugated and goat
polyclonal anti-mouse Ig Peroxidase-conjugated.
The samples were from eight different experiments, loaded as below, L-R:
Protein standard ladder Three different
experimental
conditions, A, B, C
Each condition has
control and treated –
Condition A (Control)
&
Condition A (Treated)
Two separate samples
were run for each –
A (sample 1) &
A (sample 2)
Condition A (sample 1) Control
Condition A (sample 2) Control
Condition B (sample 1) Control
Condition B (sample 2) Control
Condition C (sample 1) Control
Condition C (sample 2) Control
Condition A (sample 1) Treated
Condition A (sample 2) Treated
Condition B (sample 1) Treated
Condition B (sample 2) Treated
Condition C (sample 1) Treated
Condition C (sample 2) Treated
Protein X
What do these blots tell you about the experimental results and comment on any technical issues. (1-2
sentences per blot)
Blot 1
Blot 2
Blot 3
Blot 4
Blot 5
Blot 6
6 Shown below are two FACS outputs
For each, answer the question above the diagram
(a) The output below is of a FACS experiment to determine apoptotic state in cells treated with a
drug as follows:
• Upper Left Unstained
• Upper Right Control Cells
• Lower Left 24 h exposure to drug
• Lower Right 48h exposure to drug
Percentage of cells in each quadrat is shown in red in corner
A marker of membrane permeability is shown on the y-axis and a marker for phosphatidyl serine (an
early event in apoptosis) is shown on the x-axis
(i) What do these outputs tell you about the experiment?
(ii) Why are there no cells in the upper left quadrat in any of these outputs?
(iii) Would you expect to see a population appear there with time?
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