Question Worksheet for Cell Culture and Western Blot Virtual Experiment(2) PDF

Preview

Summary

lab...

Description

Your Name: _________________________

Lab section (day & time: ________________

BIOL 200L: Cell Biology Lab

Spring 2020

Questions for Cell Culture & Western Blot Virtual Experiment

Instructions: Answer the 25 questions based on your review of the Cell Culture and Western Blot Virtual Experiment presentation. To do so, please note the following:  Answers to these questions are worth 30 points.  You answers are due to your lab instructor by the end of your regularly scheduled lab period during the week of April 27.  The questions in this document appear within the Virtual Experiment presentation in the same order, with the same numbering (Q1-Q25), and at the appropriate times in the presentation. The questions are based on the Virtual Experiment presentation. Therefore, it will be critical to watch the Virtual Experiment presentation in order to answer the questions correctly.  To write and submit your answers, you may either: o Type your answers directly in this Word document to save and submit. o Print out this document, hand write your answers, and send your instructor a scanned copy with your answers.

Q1: Determine the volume of NGF stock solution to add to each PC12 cell culture dish. Include your work and the volume. (To receive full credit, you must show your work.)

a. Each culture of cells contains 4 ml of growth medium. b. The NGF stock concentration is 500x. Include your work and volume Cw Vw= Cs Vs) solve for Vs (1X)(4mL)(500x)(Vs) Vs= (1x)(4ml)/500x Vs=0.008 mL Vw-Vs= 4-0.008 mLof buffer to be added to 0.008 mL concentrated to make 4 mL of 1 X.

Q2: Based on this information, predict the size of proteins within regions A and B highlighted above. Size of proteins in region A: ___-100 kD___ Size of proteins in region B: ___-37 kD___ Explain how your made your predictions.

p1

The protein standards are pre-stained with dyes that enable you to see each band. When you purchase a protein standard from a biotech company, it comes with documents like the image below indicating the size of the proteins in each band. You can see the band highlighted in red on the SDS-page gel and looking at the legend to the right you are therefore able to match up the colors shown on the SDS-page gel with the colors on the legend.

Q3: The molecular weights of of Egr1 and Actin are well-established. Egr1 is 80 kD in size; Actin is 50 kD in size. Based on that information, which set of bands (a or b) show Egr1 protein levels? Which set of bands (a or b) show Actin protein levels?

Actin is B Egr is A

Q4: Do you observe significant differences in levels of Egr1 across the samples? If so, what are the differences?

Yes, there are supposed to be 4 band lines across. The first one is not seen the second on is barely visible, the third line get a little big and the fourth line is the brightest. The band line seems to intensity as you read it from left to right, and it gets dimmer and you read it from right to left.

Q5: Do you observe significant differences in levels of Actin across the samples? If so, what are the differences?

No

Q6: Does you data provide evidence that NGF treatment affects levels of Egr1 in PC12 cells? your answer. Yes, it seems to change the intensity of the band lines. Yes

Q7: Do you see any difference in cell morphology between the untreated and NGF-treated cells? Yes

p2

Q8: If you see differences in morphology, describe the differences. Treated cells have extensions coming off of the cell, they are more clustered, and they are more abundant. Whereas untreated cells are lesser in number, spaced out, and do not have any extensions coming off of the cell.

Q9: Based on your data and the background information at the beginning of this presentation, can you form a simple hypothesis describing the effect of NGF on PC12 cells? Please do so.

If PC12 cells are treated with NGF they will

e.

Q10: Does the NGF-induced morphological change have any correlation with changes in Egr1 protein levels that you observed during the Western blot analysis? Explain.

Yes, the NGF

in PC12 cells

Remember that Q11-Q16 refer to the cell counting and viability calculations for the Untreated cells. Q11: How many total cells are there in the five squares that you should examine to conduct the cell count? Square 1 : 20 live 4 dead 2 : 14 live 5 dead 3: 17 live 5 dead 4: 19 live 5 dead 5 20 live 4 dead total 113

Q12: How many live cells are there in the five squares? 90

p3

Q13: How many dead cells are there in the five squares? 23

Q14: What is the concentration of total cells (cells/ml) in the Untreated cell suspension? (You must show your work to receive full credit.)

113x 2/5 x 10,000= 452000

Q15: What is the concentration of live cells (cells/ml) in the Untreated cell suspension? (You must show your work to receive full credit.)

90x 2/5 x 10,000= 360000

Q16: What percentage of cells in the Untreated cell culture were viable? (You must show your work to receive full credit.) 90/113 x100= 79.64

360000/452000 x 100= 79.64

Remember that Q17-Q22 refer to the cell counting and viability calculations for the NGF-treated cells. Q17: How many total cells are there in the five squares that you should examine to conduct the cell count?

1: 19 live 1 dead 2: 22 live 2 dead

p4

3: 20 live 1 dead 4: 23 live 1 dead 5: 20 live 2 dead 111

Q18: How many live cells are there in the five squares?

104 Q19: How many dead cells are there in the five squares? 7 Q20: What is the concentration of total cells (cells/ml) in the NGF-treated cell suspension? (You must show your work to receive full credit.)

111x 0.4x10000= 444000

Q21: What is the concentration of live cells (cells/ml) in the NGF-treated cell suspension? (You must show your work to receive full credit.)

104x0.4x10000= 416000

Q22: What percentage of cells in the NGF-treated cell culture were viable? (You must show your work to receive full credit.)

104/111x100= 93.69

p5

Q23: Based on your calculations from the Untreated cells and NGF-treated cultures, what can you say about their relative cell viability between them? In other words, were there more or less viable cells in one of the conditions?

There were more viable cells in the NGF treated cultures

Q24: Do these observations suggest anything about the effect of NGF on PC12 cell viability? Explain.

Yes , NGF gives you more viable cells

Q25: Lastly, a fundamental practice for scientists is the consider multiple pieces of data (like you did in this experiment) it in the context of pre-existing knowledge to develop a logical working model or hypothesis. Therefore, to conclude, compose a working model or hypothesis that takes into account all of your data for: 1. 2. 3. 4.

The effect of NGF on Egr1 expression The effect of NGF PC12 cell morphology The effect of NGF on PC12 cell viability The pre-existing knowledge indicating that NGF activates the Ras/Raf/MEK/ERK signaling pathway in PC12 cells

When NGF is used , the vitality for PC12 cells will increase, the morphology of cells will show more neuron like extensions, the s across the SDS-page gel will intensify as you read it from left to right, while it gets dimmer when you read it from right to left and NGF will activate TrKA receptors which then activates the Ras/Raf/MEK/ERK signaling.

p6...