Lecture 10: SDS Page and Native Gels Analysis

Lecture Overview

• Greeting to the class
• Acknowledgement of in-person and online students
• Mention of video views from previous lectures
• Introduction to Lecture 10
  • Content Scope: 12 lectures total, focused on SDS Page and upcoming Western Blot
• Encouragement to work on homework and assignments

Analysis of SDS Page

• Emphasis on understanding data analysis from SDS Page results
• Importance of application beyond identifying band sizes
• SDS Page as a crucial tool in protein research

Terminology in SDS Page

• Pluses/Minuses: Indicates presence or absence of a protein
• Triangle/Ramp: Indicates increasing concentration
• Positive and Negative Controls: Essential for ensuring test validity
  • Example of controls and their importance
• Loading Controls: Ensure consistency across lanes
• Mutation Analysis: Detects effects of mutations (e.g. deletion, substitutions)

Example Analysis of Gel Data

• Example gel interpretation:
  • Lane with CDK1 and Cyclin B1 data
  • Delta N2 denotes deletion of N2 domain
  • S396A denotes serine to alanine substitution affecting CDK interaction
• Consistency in loading controls demonstrated with P53 and histone protein
• Example of poor loading control highlighted

Techniques of Gel Manipulation

• Discussed acceptable techniques for adjusting gels:
  • Consistent alterations to contrast settings
  • Cropping gel images for clarity
• Cropping to remove excess dead space in publications

Chromatography and SDS Page Combined

• Discussion on chromatographic techniques and their correlation with SDS Page
  • Example involving calpain chromatography and subsequent SDS Page analysis
  • Explanation of purification processes using peaks from chromatograms

Protein Analysis Examples

• Example from research on protein structure and nickel homeostasis in Helicobacter pylori:
  • Importance of histidine in binding nickel
  • Methods used for heat and confirmation analysis
• Notion of molecular weight calculations determining bound nickel count

Case Study: MEF2C Analysis

• Title: "Phosphorylation Facilitated Sumulation of MEF2C"
• MEF2C: A key player in gene expression and muscle development
  • Describes the impact of phosphorylation on sumoylation
• Critical positions in MEF2C: lysine 391 and serine 396
• Study insights:
  • Alignment of various myocyte enhancers
  • Key mutations and their observed effects on sumoylation necessity
• Labels used to denote wild type versus mutant configurations

Experimental Results

• Display of experiments demonstrating the attachment of SU(MO) groups on MEF2C
• Elucidated findings on lysine versus arginine substitutions affecting sumoylation
  • Importance of specific residues in protein modifications
• Observed mutations impacting phosphorylation and their implications on transcriptional activity

Lecture Continuation: Native Gels

• Introduction to native gels, isoelectric focusing, and 2D gel electrophoresis
  • Explanation of native (non-denaturing) gels retaining protein structures
• Migration influences and protein activity on native gels versus SDS Page
• Detailed explanation of the ionic environment and charge-based migration

Applications of Native Gels

• Determining conformation and oligomeric states of proteins
• Analysis methods available detectable in native gels
• Enabling protein interactions studies, enzyme assays, and activity maintenance

Comparison between Native Gels and SDS Page

• Molecular weight resolution limitations on native gels
  • Examples of difficulty correlating with SDS Page results
• Importance of charge and size variations in mobility within native gels
• Real-world applications and limitations in experimental design with native gels

Experimental Procedures for Protein Characterization

• Different methodologies outlined for assessing protein properties:
  • Phosphorylation, glycosylation, and structural assessments using native gels
• Examples referenced in literature concerning structural and functional analysis of glycoproteins

Hybrid Experiments and Interpretations

• Case studies showcasing hybrid experiments with unique proteins, addressing interaction implications and studying their post-translational modifications
• Relationship between native graphs and bands distilling critical conclusions in protein research

Summary and Conclusion

• MSAT questions provided highlighting key focuses on SDS Page and native gels, ensuring comprehension in concepts discussed
• Focus on implications in biological research, emphasizing integrative methods across diverse gel techniques
• Closing remarks inviting students to apply understanding in practical experiments
• Final thoughts ahead of next session regarding continued studies in gel electrophoresis techniques.