BIOL3380 Section 101 (R-AM) Experiment 7 Western Blot Analysis of rGFP Fractions<University of Texas, Dallas - BIOL 3380>Lab Report 7

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Jessica Nguyen BIOL3380 Section 101 (R-AM) Experiment 7 Western Blot Analysis of rGFP Fractions Partner: Victoria Tran Graduate TA: Lalitha Kumar 1. You will receive this credit if you perfor... med the SDS-PAGE gel and set up the Western Transfer. 2. What is the general purpose of a Western Blot? 3. Ponceau S is a red dye that reversibly binds to all proteins. We will use this stain prior to developing the Western Blot next week with antibodies. Assume that you do not observe any protein bands on their nitrocellulose blot after performing the Ponceau S Stain. Describe one likely procedural error in the transfer set-up that would account for this observation. 4. Why will we use antibodies that recognize the X-press epitope tag and not the His6 tag? 5. Chromatography columns have a limited protein binding capacity. During lab #4 you should not have “overloaded” the Ni+2 Agarose column. Yet you should have noticed that the W2 fraction fluoresced slightly. Based upon our Western Blot antibody procedure, would you expect to see a band in the W2 lane? If so, what is it’s MW? What does this expected data say about the physical protein structure of rGFP? 6. If you did “overload” your Ni+2 Agarose column with a GCE sample that contained too much rGFP, would you expect to see a band in the W2 lane of the Western Blot? If so, what is it’s MW? Would you expect to see a band in the E2 lane? If so, what’s it’s MW? 7. When you develop the Western Blot, what MW size band in lane E3 are you expecting to observe? If you happen to see two bands – one at the expected MW and one lower band – what might you specifically conclude about the physical protein structure of the lower band? [Show More]

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