Problem Based Learning Problem-Solving Test: Southwestern Blotting
nyi* zsef Szebere Jo
From the Department of Medical Biology, Medical School, University cs, H-7624 Pe cs, Hungary of Pe
Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA, deletion mutants, expression plasmid, transfection, RNA polymerase II, promoter, ShineDalgarno sequence, polyadenylation element, affinity chromatography, Northern blotting, immunoprecipitation, sodium dodecylsulfate, autoradiography, tandem repeats
The Experiment The term “Southwestern blotting” is a word-wit: it combines the names of two widely used molecular biology techniques, Southern blotting and Western blotting (or immunoblotting). Refresh your memories and solve the following multiple-choice questions (MCQs) related to the two blotting methods.
Four-choice Association (In this type of question a set of lettered headings is followed by a list of numbered words or phrases. Select: A. B. C. D.
if the word or phrase is associated with A only; if the word or phrase is associated with B only; if the word or phrase is associated with A and B; if the word or phrase is associated with neither A nor B.)
Abbreviations: MCQ, multiple-choice question; PAGE, polyacrylamide gel electrophoresis; SH, Src-homology; NLS, nuclear localization signal; SDS, sodium dodecylsulfate *Address for correspondence to: Department of Medical Biology, Med cs, H-7624 Pe cs, Hungary. E-mail: ical School, University of Pe [email protected]. Received 10 June 2014; Accepted 15 July 2014 DOI 10.1002/bmb.20816 Published online 5 August 2014 in Wiley Online Library (wileyonlinelibrary.com)
Biochemistry and Molecular Biology Education
A. B. C. D.
Southern blotting Western blotting Both of them Neither of them
1.____Restriction endonuclease-generated DNA fragments are fractionated by agarose gel electrophoresis in this method. 2.____A nitrocellulose filter can be used for the transfer of macromolecules from the gel. 3.____Detection of specific target molecules is achieved by molecular hybridization. 4.____Can be used to identify DNA–protein interactions. 5.____Specific antibodies are used in this technique to identify the target molecules. 6.____A step in this method is the fractionation of proteins by polyacrylamide gel electrophoresis (PAGE). Southwestern blotting combines some of the elements of these two techniques, with a completely different aim. The method was developed by Bowen et al. [1]. The example used to present the principle of the technique in this test is the characterization of an important protooncogenic protein, c-Abl [2]. (A problem-solving test dealing with the features of a mutationally activated, leukemogenic form of c-Abl was published earlier in this series [3].) The domain structure of the c-Abl protein is shown in Fig. 1a. Its N-terminal region contains the Src-homology (SH) domains SH3, SH2, and SH1 (the latter corresponds to the tyrosine protein kinase domain). The C-terminal part of the protein contains nuclear localization signals (NLS), but otherwise is functionally less well characterized. Full length cDNA (c-abl) coding for the protein was synthesized and deletion mutants (DSal, DXho-Sal, DApa-Apa) were generated with the restriction endonucleases shown in Fig. 1a. The cDNA constructs were inserted into expression plasmids, transfected into suitable mammalian host cells, and the c-Abl proteins were isolated from the cell cultures.
Five-choice Completion (This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)
443
Biochemistry and Molecular Biology Education The four purified protein samples were run in two parallel sodium dodecylsulfate (SDS)-polyacrylamide gels. The gels were soaked in a buffer after electrophoresis to remove SDS (this leads to the renaturation of the proteins in their native conformation). The proteins in both gels were transferred to nitrocellulose filters. One membrane was incubated with 32P-labeled genomic DNA fragments and bound molecules were detected by autoradiography (Fig. 1b, left panel). Western blotting was performed on the other membrane using an anti-c-Abl antibody (Fig. 1b, right panel). Study the figure and solve the following MCQs.
Figure Analysis (The following statements are related to the information presented above. Based on the information given Select:
FIG 1
Analysis of c-Abl and its truncated forms by Southwestern blotting. (a) The domain structure of c-Abl protein, restriction endonuclease cleavage sites on c-abl cDNA, and the structure of deletion constructs used in this study. (b) Analysis of c-Abl proteins by Southwestern blotting and immunoblotting (for experimental details see the text; § and 丣 indicate the position of electrodes during electrophoresis).
7.____Which of the following sequences should be present in a mammalian expression plasmid for efficient expression of the protein? A. RNA polymerase II promoter B. Shine-Dalgarno sequence C. Polyadenylation element D. A and C E. A, B, and C 8.____Which of the following techniques would be suitable for the isolation of the c-Abl proteins from cellular extracts? A. Affinity chromatography on an anti-c-Abl-antibodyagarose column B. Northern blotting using a c-abl specific probe C. Immunoprecipitation using anti-c-Abl antibody D. A and C E. A, B, and C
444
A. if the statement is supported by the information given; B. if the statement is contradicted by the information given; C. if the statement is neither supported nor contradicted by the information given.) 9.____The peptide region encoded by the XhoI-SalI cDNA region is essential for the stabilization of the c-Abl protein. 10.____Autophosphorylation of the c-Abl protein is required for DNA binding. 11.____The SH3 domain is required for DNA binding. 12.____The SH3 domain is sufficient to provide stable DNA binding. 13.____The antibody used in this experiment recognized the c-Abl epitope that corresponds to the XhoI-ApaI region. 14.____The four c-Abl-derived polypeptides were separated during electrophoresis according to their size. 15.____The c-Abl protein recognizes and binds to specific sequences in DNA. 16.____The peptide region corresponding to the XhoI-ApaI cDNA fragment contains the DNA binding domain. 17.____The XhoI-ApaI fragment corresponds to an intron.
Five-choice Completion (This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.) 18.____Based on the experiment described above determine the aim of using Southwestern blotting: A. B. C. D. E.
Detection of protein–protein interactions Detection of DNA–protein interactions Identification of tandemly repeated sequences in DNA Identification of specific nuclear proteins Identification of specific cytoplasmic proteins
Southwestern Blotting
Correct Answers 1.
A
10. C
2.
C
11. C
3.
A
12. B
4.
D
13. B
5.
B
14. A
6.
B
15. C
7.
D
16. A
8.
D
17. B
9.
B
18. B
Explanations Southern blotting is used to identify specific restriction endonuclease fragments by hybiridizing the membrane blot to a specific labeled nucleic acid probe (MCQ 1: A; MCQ 3: A). In Western blotting a protein is identified among proteins fractionated by PAGE and transferred to a membrane, using the antibody specific for the protein (MCQ 2: C; MCQ 4: D; MCQ 5: B; MCQ 6: B). Southwestern blotting is used to analyze DNA-binding proteins (MCQ 18: B). The expression vector used to express the c-Abl proteins in mammalian cells needs to contain a PolII promoter
Szeberenyi
and a polyadenylation element upstream and downstream, respectively, of the inserted cDNA fragment (MCQ 7: D; the Shine-Dalgarno sequence is a ribosome binding site of prokaryotic mRNAs). The c-Abl proteins can be purified from cellular extracts by immunoprecipitation or affinity chromatography (MCQ 8: D). c-Abl is a DNA-binding protein. Southwestern blotting of the c-Abl proteins fractionated by size indicated that the DNA-binding domain is in the C-terminal region encoded by an exon of the XhoI-ApaI fragment (MCQ 12: B; MCQ 14: A; MCQ 16: A; MCQ 17: B). The DXho-Sal c-Abl protein is as stable as the other constructs (see the immunoblot in Fig. 1b, MCQ 9: B). The antibody recognized all four c-Abl proteins (see the immunoblot in Fig. 1b) and thus was probably raised against the N-terminal part of the protein (MCQ 13: B). The role of the phosphorylation state, SH3 domain, and the sequence-specificity of DNA-binding could not be judged from this experiment (MCQ 10: C; MCQ 11: C; MCQ 15: C).
References [1] Bowen B., Steinberg, J., Laemmli, U. K., Weintraub, H. (1980) The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 8, 1– 20. [2] Kipreos E. T. and Wang, J.Y.J. (1992) Cell cycle-regulated binding of cAbl tyrosine kinase to DNA. Science 256, 382–385. nyi J. (2012) Problem-solving test: Signal transduction in Phila[3] Szebere delphia chromosome positive leukemia cells. Biochem. Mol. Biol. Educ. 40, 333–336.
445
nyi* zsef Szebere Jo
From the Department of Medical Biology, Medical School, University cs, H-7624 Pe cs, Hungary of Pe
Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA, deletion mutants, expression plasmid, transfection, RNA polymerase II, promoter, ShineDalgarno sequence, polyadenylation element, affinity chromatography, Northern blotting, immunoprecipitation, sodium dodecylsulfate, autoradiography, tandem repeats
The Experiment The term “Southwestern blotting” is a word-wit: it combines the names of two widely used molecular biology techniques, Southern blotting and Western blotting (or immunoblotting). Refresh your memories and solve the following multiple-choice questions (MCQs) related to the two blotting methods.
Four-choice Association (In this type of question a set of lettered headings is followed by a list of numbered words or phrases. Select: A. B. C. D.
if the word or phrase is associated with A only; if the word or phrase is associated with B only; if the word or phrase is associated with A and B; if the word or phrase is associated with neither A nor B.)
Abbreviations: MCQ, multiple-choice question; PAGE, polyacrylamide gel electrophoresis; SH, Src-homology; NLS, nuclear localization signal; SDS, sodium dodecylsulfate *Address for correspondence to: Department of Medical Biology, Med cs, H-7624 Pe cs, Hungary. E-mail: ical School, University of Pe [email protected]. Received 10 June 2014; Accepted 15 July 2014 DOI 10.1002/bmb.20816 Published online 5 August 2014 in Wiley Online Library (wileyonlinelibrary.com)
Biochemistry and Molecular Biology Education
A. B. C. D.
Southern blotting Western blotting Both of them Neither of them
1.____Restriction endonuclease-generated DNA fragments are fractionated by agarose gel electrophoresis in this method. 2.____A nitrocellulose filter can be used for the transfer of macromolecules from the gel. 3.____Detection of specific target molecules is achieved by molecular hybridization. 4.____Can be used to identify DNA–protein interactions. 5.____Specific antibodies are used in this technique to identify the target molecules. 6.____A step in this method is the fractionation of proteins by polyacrylamide gel electrophoresis (PAGE). Southwestern blotting combines some of the elements of these two techniques, with a completely different aim. The method was developed by Bowen et al. [1]. The example used to present the principle of the technique in this test is the characterization of an important protooncogenic protein, c-Abl [2]. (A problem-solving test dealing with the features of a mutationally activated, leukemogenic form of c-Abl was published earlier in this series [3].) The domain structure of the c-Abl protein is shown in Fig. 1a. Its N-terminal region contains the Src-homology (SH) domains SH3, SH2, and SH1 (the latter corresponds to the tyrosine protein kinase domain). The C-terminal part of the protein contains nuclear localization signals (NLS), but otherwise is functionally less well characterized. Full length cDNA (c-abl) coding for the protein was synthesized and deletion mutants (DSal, DXho-Sal, DApa-Apa) were generated with the restriction endonucleases shown in Fig. 1a. The cDNA constructs were inserted into expression plasmids, transfected into suitable mammalian host cells, and the c-Abl proteins were isolated from the cell cultures.
Five-choice Completion (This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)
443
Biochemistry and Molecular Biology Education The four purified protein samples were run in two parallel sodium dodecylsulfate (SDS)-polyacrylamide gels. The gels were soaked in a buffer after electrophoresis to remove SDS (this leads to the renaturation of the proteins in their native conformation). The proteins in both gels were transferred to nitrocellulose filters. One membrane was incubated with 32P-labeled genomic DNA fragments and bound molecules were detected by autoradiography (Fig. 1b, left panel). Western blotting was performed on the other membrane using an anti-c-Abl antibody (Fig. 1b, right panel). Study the figure and solve the following MCQs.
Figure Analysis (The following statements are related to the information presented above. Based on the information given Select:
FIG 1
Analysis of c-Abl and its truncated forms by Southwestern blotting. (a) The domain structure of c-Abl protein, restriction endonuclease cleavage sites on c-abl cDNA, and the structure of deletion constructs used in this study. (b) Analysis of c-Abl proteins by Southwestern blotting and immunoblotting (for experimental details see the text; § and 丣 indicate the position of electrodes during electrophoresis).
7.____Which of the following sequences should be present in a mammalian expression plasmid for efficient expression of the protein? A. RNA polymerase II promoter B. Shine-Dalgarno sequence C. Polyadenylation element D. A and C E. A, B, and C 8.____Which of the following techniques would be suitable for the isolation of the c-Abl proteins from cellular extracts? A. Affinity chromatography on an anti-c-Abl-antibodyagarose column B. Northern blotting using a c-abl specific probe C. Immunoprecipitation using anti-c-Abl antibody D. A and C E. A, B, and C
444
A. if the statement is supported by the information given; B. if the statement is contradicted by the information given; C. if the statement is neither supported nor contradicted by the information given.) 9.____The peptide region encoded by the XhoI-SalI cDNA region is essential for the stabilization of the c-Abl protein. 10.____Autophosphorylation of the c-Abl protein is required for DNA binding. 11.____The SH3 domain is required for DNA binding. 12.____The SH3 domain is sufficient to provide stable DNA binding. 13.____The antibody used in this experiment recognized the c-Abl epitope that corresponds to the XhoI-ApaI region. 14.____The four c-Abl-derived polypeptides were separated during electrophoresis according to their size. 15.____The c-Abl protein recognizes and binds to specific sequences in DNA. 16.____The peptide region corresponding to the XhoI-ApaI cDNA fragment contains the DNA binding domain. 17.____The XhoI-ApaI fragment corresponds to an intron.
Five-choice Completion (This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.) 18.____Based on the experiment described above determine the aim of using Southwestern blotting: A. B. C. D. E.
Detection of protein–protein interactions Detection of DNA–protein interactions Identification of tandemly repeated sequences in DNA Identification of specific nuclear proteins Identification of specific cytoplasmic proteins
Southwestern Blotting
Correct Answers 1.
A
10. C
2.
C
11. C
3.
A
12. B
4.
D
13. B
5.
B
14. A
6.
B
15. C
7.
D
16. A
8.
D
17. B
9.
B
18. B
Explanations Southern blotting is used to identify specific restriction endonuclease fragments by hybiridizing the membrane blot to a specific labeled nucleic acid probe (MCQ 1: A; MCQ 3: A). In Western blotting a protein is identified among proteins fractionated by PAGE and transferred to a membrane, using the antibody specific for the protein (MCQ 2: C; MCQ 4: D; MCQ 5: B; MCQ 6: B). Southwestern blotting is used to analyze DNA-binding proteins (MCQ 18: B). The expression vector used to express the c-Abl proteins in mammalian cells needs to contain a PolII promoter
Szeberenyi
and a polyadenylation element upstream and downstream, respectively, of the inserted cDNA fragment (MCQ 7: D; the Shine-Dalgarno sequence is a ribosome binding site of prokaryotic mRNAs). The c-Abl proteins can be purified from cellular extracts by immunoprecipitation or affinity chromatography (MCQ 8: D). c-Abl is a DNA-binding protein. Southwestern blotting of the c-Abl proteins fractionated by size indicated that the DNA-binding domain is in the C-terminal region encoded by an exon of the XhoI-ApaI fragment (MCQ 12: B; MCQ 14: A; MCQ 16: A; MCQ 17: B). The DXho-Sal c-Abl protein is as stable as the other constructs (see the immunoblot in Fig. 1b, MCQ 9: B). The antibody recognized all four c-Abl proteins (see the immunoblot in Fig. 1b) and thus was probably raised against the N-terminal part of the protein (MCQ 13: B). The role of the phosphorylation state, SH3 domain, and the sequence-specificity of DNA-binding could not be judged from this experiment (MCQ 10: C; MCQ 11: C; MCQ 15: C).
References [1] Bowen B., Steinberg, J., Laemmli, U. K., Weintraub, H. (1980) The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 8, 1– 20. [2] Kipreos E. T. and Wang, J.Y.J. (1992) Cell cycle-regulated binding of cAbl tyrosine kinase to DNA. Science 256, 382–385. nyi J. (2012) Problem-solving test: Signal transduction in Phila[3] Szebere delphia chromosome positive leukemia cells. Biochem. Mol. Biol. Educ. 40, 333–336.
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