PAGE and Western Blot Practice Sheet_Answer Key

pdf

School

University of Houston *

*We aren’t endorsed by this school

Course

3201

Subject

Biology

Date

Apr 3, 2024

Type

pdf

Pages

Uploaded by SuperBravery6984

BCHS3201 Polyacrylamide Gels and Western Blots Practice Sheet 1. In the gels pictured here, lane 1 is a protein molecular weight marker (values are in kDa). Lane 2 is the Reallyfascinating protein. Assuming this protein is composed of 2 subunits, what is the molecular weight of this protein? 82 kDa 2. Dr. Pattison is working with the NotActuallyReal protein which is composed of the following subunits: A (42 kDa), two subunits of B (16 kDa) and C (30 kDa). Subunits A and C are tethered by a disulfide bond. a. How many bands would you see in a native gel? one b. On the gel picture below, draw the band pattern in an SDS denaturing gel in the first blank lane. Bands: 42 kDa, 16 kDa, 30 kDa SDS PAGE Native 1 1 2 2

c. In the second blank lane, draw the pattern if Dr. Pattison was distracted by a question from a lab member and forgot to add beta-mercaptoethanol to the sample. Bands 72 kDa and 16 kDa d. If subunit C was post-translationally glycosylated, how would this impact migration on the SDS-gel? Subunit C would migrate at a higher apparent molecular weight than expected. 3. Dr. Pattison has grown a culture of E.coli that has been induced to overexpress the Drosophiola melanogaster ultrabithorax protein (47.5 kDa) for use in the BCHS4311 Biochem 2 Lab course. After allowing the culture to grow overnight, she spins it down and lyses the cells. a. Sketch what she would see on the gel in the first blank lane below if she runs the lysate on an SDS-PAGE gel and stains with Coomassie Blue. b. Is this sufficient to demonstrate that the ultrabithorax protein has been successfully overexpressed in this cell line? No, there could be another 47.5 kDa protein or one very close in size that is expressed at a high level but is not the ultrabithorax protein. There is no way to distinguish between the two possibilities on a Coomassie stained gel.

c. Dr. Pattison runs another gel with the lysate sample and this time transfers the proteins to a nylon membrane. She conducts a Western blot with antibodies against the ultrabithorax protein. Sketch would she would see on the blot in second blank lane below. One band at 47.5 kDa d. If Dr. Pattison forgot to add blocking agent to the pre-hybridization buffer, sketch would she would see after developing the blot in the third blank lane on the gel below. Probe would stick everywhere so the entire blot would be developed, making it difficult to impossible to see the targeted protein band.

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Related Documents

2_Proj_S23.docx

4_Proj_S23.docx

8_Proj_S23.docx

Projects Six and Seven.docx

Zoology Lab Tutorial Assignment.docx

MCCG150 Week 5 Initial Discussion post.docx

EXAM 3 - practice .pdf

Copy of INFO 4 Mutation analysis problem KEY.docx

Practice Questions.pdf

Practice Questions.docx

Copy of INFO 4 Transcription and translation application problem and KEY.docx

Culinary Fundamentals HW #18.docx

Recommended textbooks for you

Human Heredity: Principles and Issues (MindTap Co...

Biology

ISBN:9781305251052

Author:Michael Cummings

Publisher:Cengage Learning

Biochemistry

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Cengage Learning

Biology: The Dynamic Science (MindTap Course List)

Biology

ISBN:9781305389892

Author:Peter J. Russell, Paul E. Hertz, Beverly McMillan

Publisher:Cengage Learning

Biochemistry

ISBN:9781305961135

Author:Mary K. Campbell, Shawn O. Farrell, Owen M. McDougal

Publisher:Cengage Learning

Biology 2e

Biology

ISBN:9781947172517

Author:Matthew Douglas, Jung Choi, Mary Ann Clark

Publisher:OpenStax

Basic Clinical Laboratory Techniques 6E

Biology

ISBN:9781133893943

Author:ESTRIDGE

Publisher:Cengage

SEE MORE TEXTBOOKS

Recommended textbooks for you

Human Heredity: Principles and Issues (MindTap Co...
Biology
ISBN:9781305251052
Author:Michael Cummings
Publisher:Cengage Learning
Biochemistry
Biochemistry
ISBN:9781305577206
Author:Reginald H. Garrett, Charles M. Grisham
Publisher:Cengage Learning
Biology: The Dynamic Science (MindTap Course List)
Biology
ISBN:9781305389892
Author:Peter J. Russell, Paul E. Hertz, Beverly McMillan
Publisher:Cengage Learning
Biochemistry
Biochemistry
ISBN:9781305961135
Author:Mary K. Campbell, Shawn O. Farrell, Owen M. McDougal
Publisher:Cengage Learning
Biology 2e
Biology
ISBN:9781947172517
Author:Matthew Douglas, Jung Choi, Mary Ann Clark
Publisher:OpenStax
Basic Clinical Laboratory Techniques 6E
Biology
ISBN:9781133893943
Author:ESTRIDGE
Publisher:Cengage