Lab #7 ELN Weekly Guidelines - SDS-PAGE and Transfer
Now that we have successfully created and cloned our pHLsec-CaMPARI, we are now
transitioning to studying a new vector: pET41a-CaMPARI. pET41a is a BACTERIAL EXPRESSION
vector that also has a GST tag at the 3’ end of CaMPARI. The theme of these next labs is to isolate and
visualize our CaMPARI protein (made possible by the GST tag) and understand how CaMPARI
functions as a calcium indicator. Once we are sure that our CaMPARI protein is able to successfully
detect changes in calcium, we can finally move on to transfecting (aka ‘transforming’ but for
mammalian cells) our pHLsec-CaMPARI vector into mammalian cells and perform our calcium
detection experiment on cancer drugs applied to our Chinese Hamster Ovary cells.
PURPOSE
❏ Briefly mention ALL protocols that will be used and how these protocols will help you reach the
CaMPARI project goals. Be specific with what samples are involved in what protocols.
❏ What are we doing this week and how are we doing it?
❏ Why are we using pET41a? What cells are we using? Why are these cells being treated
with Lactose and IPTG?
❏ Why are we doing the experiments this week and how do the results fit into the overall
CaMPARI workflow?
❏ What is an inducible expression vector and why is this important to the workflow?
METHODS
Copy and paste the necessary protocols
RESULTS – Figures & Tables
❏ Annotated SDS-PAGE gel figure with complete legend.
❏ Legend description should include information about the gel (Percentage, Amperage, Time,
etc.) and the samples loaded in each of the gel lanes (include sample names and blank wells).
❏ Should include the specific name of the Protein ladder, the type and concentration of
induction of each sample
❏ Make it clear what the control is
❏ Annotated Membrane figure (after transfer of gel) with complete legend
❏ Legend should have same sample details but different title
RESULTS – Text (For each of these figures you should include textual results in paragraph form)
❏ You should include what controls were used and how the experimental results compare to the controls.
❏ Regions of interest: GST:CaMPARI (78.4 kDa) and GST (25.1 kDa)
DISCUSSION
(This should be a discussion of your results and how they fit into the CaMPARI workflow)
❏ Do you suspect CaMPARI is present, even though we cannot prove it until we perform our Western
Blot? Why are there many other bands in SDS-PAGE?
❏ Can you be confident your Western Blot will work based on SDS-PAGE gel? Why or why not?
❏ What type of induction (lactose or IPTG) do you PREDICT will produce the most CaMPARI protein and
why? [HINT: Read the introduction section of the protocols. We will find out for sure when we quantify
the darkness of our Western Blot bands]
DISCUSSION QUESTIONS
Now that we have successfully created and cloned our pHLsec-CaMPARI, we are now
transitioning to studying a new vector: pET41a-CaMPARI. pET41a is a BACTERIAL EXPRESSION
vector that also has a GST tag at the 3’ end of CaMPARI. The theme of these next labs is to isolate and
visualize our CaMPARI protein (made possible by the GST tag) and understand how CaMPARI
functions as a calcium indicator. Once we are sure that our CaMPARI protein is able to successfully
detect changes in calcium, we can finally move on to transfecting (aka ‘transforming’ but for
mammalian cells) our pHLsec-CaMPARI vector into mammalian cells and perform our calcium
detection experiment on cancer drugs applied to our Chinese Hamster Ovary cells.
PURPOSE
❏ Briefly mention ALL protocols that will be used and how these protocols will help you reach the
CaMPARI project goals. Be specific with what samples are involved in what protocols.
❏ What are we doing this week and how are we doing it?
❏ Why are we using pET41a? What cells are we using? Why are these cells being treated
with Lactose and IPTG?
❏ Why are we doing the experiments this week and how do the results fit into the overall
CaMPARI workflow?
❏ What is an inducible expression vector and why is this important to the workflow?
METHODS
Copy and paste the necessary protocols
RESULTS – Figures & Tables
❏ Annotated SDS-PAGE gel figure with complete legend.
❏ Legend description should include information about the gel (Percentage, Amperage, Time,
etc.) and the samples loaded in each of the gel lanes (include sample names and blank wells).
❏ Should include the specific name of the Protein ladder, the type and concentration of
induction of each sample
❏ Make it clear what the control is
❏ Annotated Membrane figure (after transfer of gel) with complete legend
❏ Legend should have same sample details but different title
RESULTS – Text (For each of these figures you should include textual results in paragraph form)
❏ You should include what controls were used and how the experimental results compare to the controls.
❏ Regions of interest: GST:CaMPARI (78.4 kDa) and GST (25.1 kDa)
DISCUSSION
(This should be a discussion of your results and how they fit into the CaMPARI workflow)
❏ Do you suspect CaMPARI is present, even though we cannot prove it until we perform our Western
Blot? Why are there many other bands in SDS-PAGE?
❏ Can you be confident your Western Blot will work based on SDS-PAGE gel? Why or why not?
❏ What type of induction (lactose or IPTG) do you PREDICT will produce the most CaMPARI protein and
why? [HINT: Read the introduction section of the protocols. We will find out for sure when we quantify
the darkness of our Western Blot bands]
DISCUSSION QUESTIONS
