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PROTEIN CONCENTRATION
CHECKING RESULTS OF LIGATION/TRANSFORMATION
INQUIRY: What is the probability that your colonies would have the insert if you don’t
have blue/white screening to help you identify them? How could you estimate this?
Protein determination – Refer back to Lab Manual 3 for a description of protein many
different protein determinations that have been used through the history of biochemistry.
http://wolfson.huji.ac.il/purification/Protocols/Comparision_of_Methods.htm
The importance of the protein determination in our purification of the LDH enzymes
(beef heart and barracuda) is that it enables us to calculate the specific activity of the
LDH. The specific activity (SA – product formed / unit time / mg protein) is our ultimate
measure of purification of active enzyme. To make your purification table, you need to
determine the amount of protein in samples you took at each step of the purification,
and then back calculate the total protein in each step. This is the total (bulk) protein, of
which, as you go through the purification will contain a larger and larger fraction of LDH.
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BioC 4025W Fall 2017 Lab 7
The blue white selection (see week 6 lecture and prelab question) also allows you to
know if your insert was ligated into the vector.
How can you select for cells that have the plasmid?
How can you screen for the plasmid containing the insert?
The plasmid vector you used had Blue/White (B/W) test capability and confirms Ampicillin
(Amp)-resistant. The E. coli cells are also engineered to be B/W capable. Set up and plate 6
transformations (3 ligation rxns and 3 controls).
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BioC 4025W Fall 2017 Lab 7
Controls
Transformed with Competent cells Competent cells
Circular B/W plasmid +Amp no Amp
IPTG + X-gal +Amp
Assuming that your experiment worked, describe or draw what you see on each plate. If you
draw, use open circles [] for white colonies and filled circle [] for blue colonies. Qualitative
answers are sufficient (not quantitative).
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BioC 4025W Fall 2017 Lab 7
Procedure 1
FIRST THING,if you did size exclusion, you would do an activity assay on your conc’t
SEC—Instead, do an activity assay on your conc’t AFC! This is also a good time to re-
assay any sample that you don’t have an activity assay data.
Protein Concentrations
Standard Curve
b. Set up 3 ml cuvettes.
Reagent/Cuvette 1 2 3 4 5 6 7 8
BSA Standard, 1 mg/ml (µl) 0 10 20 30 40 50 75 100
Bradford reagent 3 ml →
Pour about 100 ml of BRADFORD REAGENT INTO THE 250 ml GLASS BEAKER at
your station.
NOTE: DO NOT put in plastic as it stains! Your plastic beakers have been
removed so they cannot be contaminated with Bradford Reagent. Put tips into the
large plastic beaker you share with the group across from you.
Reagent/Cuvette 1 2 3 4 5 6 7 8 etc.
Sample (µl) [see table next page]
Bradford reagent 3 ml →
Use 10 µl of your samples unless they are extremely “gunky” e.g. the crude sample.
You will need to dilute those samples 1 - 10 then use 10 µl of that diluted sample for
your assay. Be sure to account for the dilution in your calculations. Dilute with
water.
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BioC 4025W Fall 2017 Lab 7
2. Add 3 ml Bradford reagent to each cuvette and mix well immediately. Do not wait
unit you have added it to all cuvettes.
3. Incubate at least 10 minutes at RT. Once the color develops, it is stable for over
an hour. While it sits, now is a good time to peer evaluate LLR writing samples of
Methods and Results. Look at the Rubric Guidelines
Remember, this chart below will go into your large lab report.
Hint: you should have done an activity on the conc’t AFC sample.
Dialyzed 65% AS
pellet (dial 65%)
Pooled Affinity
fractions (pool AFC)
Conc’t Affinity fractions
(conc’t AFC)
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BioC 4025W Fall 2017 Lab 7
Procedure 2:
Circle two colonies from plate A or B or C and put your color tape by the circled
colonies. If you don’t have any colonies on either A or B, circle two colonies from plate
C. What probability would you expect? How could you estimate based on v:i DNA ratio?
If you don’t have colonies on A, B, or C, circle two colonies from someone else in the
lab. Put your initials on your color tape by these circles.
The colonies that you circled will be used to inoculate a 5 ml culture of LB/Amp so you
can do plasmid preps next week. You will get your cultures back next lab period.
INQUIRY: Design an
Just for thought: Awexperiment
snap! Doeswith
our the his-tag
plasmid using
have thechromatography.
B/W screen? How does
the protein bind and how would you get it off?
Based on vector:insert ratios and control plates, what is the probability that the
colony you pick will have your LDH gene insert cloned into the vector?
What are three or more ways we could test this to find out?
Lab Report 7 (there was no lab report 6 so it is 6-7), including data and questions.
Prelab write-up in notebook Lab 8, Procedures 1- 5, but pre-lab question is just to watch
the video below.
https://www.khanacademy.org/test-rep/mcat/biomolecules/enzyme-kinetics/v/an-introduction-to-enzyme-kinetics
Next lab 8 is during midterm week, so be sure to view Kahn academy enzyme kinetics
in advance.
CLEAN UP: SERIOUSLY, THIS IS AN EASY ONE….CHECK OUT THE HANDOUT,
EASY BREEZY.
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