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BIOCHEM LAB EXAM 3
Terms in this set (21)
Western Blot Analysis of rGFP fractions
Confirm the presence of rGFP using specific antibodies and identify any degradation products if present
Transfer SDS gel to membrane (blot) and probe (detect)
Analysis of Blot
Full length of rGFP is 32 kD
-Partial degradation if around 28kD (below full length of rGFP)
- N-terminus: The Xpress tag is cut off
- C-terminus: The Xpress tag is not, meaning that
one can still see the band.
- Contaminant: Anything in E2 or E3 above the 32 kD band
7a Step Up:
Locking lid: Negative electrode
3 filter paper
10% resolving gel
3 filter paper
Base: Positive electrode
7b Step Up:
- Blocking agent using powdered milk (blocks unoccupied sites so that the primary and secondary antibodies will bind specifically to sites in order to localize the protein of interest to a single protein band on the membrane)
- Addition of primary antibody
(Washed with Tween 20)
- HRP-coupled secondary antibody
(Washed with Tween 20)
- Develop film
- Addition of chemicaliluminescent (TMB) substrate to its oxidized form and exposure film
A student wants to use western blotting to confirm the presence of human Sonic hedgehog (Shh) in samples run on SDS-PAGE and transfered to a nitrocellulose membrane. The antibody reagent box in their lab contains Mouse anti-Shh MAb and Chicken anti-mouse IgG-HRP. What substrate will they need in order to perform colorimetric detection of Shh on their western blot?
A student used western blotting to confirm the presence of C. elegans SMAP5 in samples run on SDS-PAGE and transferred to a nitrocellulose membrane. The image above shows all interactions prior to the addition of substrate. Which component of the image represents SMAP5?
SMAP5 (antigen) represents the black circle
A student used western blotting to confirm the presence of horse Twist2 protein in osteoblast samples run on SDS-PAGE and transfered to a nitrocellulose membrane. The top image shows all interactions prior to the addition of substrate. Which of the following antibodies did the student have the lab technician buy in order to successfully perform the western blot and what component of the image represents that antibody?
Mouse-antiTwist 2; red structure
- Explanation: Horse Twist2 is the antigen. The primary antibody needs to be anti-Twist2. Already given the secondary antibodies, the primary antibody is the red structure
A student wants to use western blotting to confirm the presence of chicken collagen type II in samples run on SDS-PAGE and transferred to a nitrocellulose membrane. If they use Mouse anti-collagen type II MAb and TMB as part of a successful western blot, which of the following could the "Green Y with purple knob" in the figure be?
Donkey anti-mouse IgG-HRP
- Explanation: Mouse anti-collagen type II MAb is the primary antibody, so secondary antibody needs to be anti-mouse IgG. Since TMB is the substrate, the enzyme needs to be HRP.
Which of the following bind proteins nonspecifically?
Which column would you use to purify a 32kd positively charged tagged protein from a 35kd negatively charged protein?
Ion exchange column bc separate based on charge
Which column would you use to purify a 175kd negatively charged tagged protein from a 220kd negatively charged protein?
G200 gel filtration column bc separate based upon size. The stated difference between the two proteins is 175kD and 220 kD, the G200 matrix will allow the 175 kD to enter the beads and exclude the 220 kD
Larger proteins elute off a Ni+2 agarose column in early fractions.
Larger proteins elute off a gel filtration column in earlier fractions.
An elution fraction from a Ni+2 agarose column that has a high rGFP florescence will also have a high purity.
A highly positive charged protein will bind a anion exchanger and elute of with a high salt buffer.
A highly positive charged protein will bind a cation exchanger and elute off by changing the pH.
How many grams of KCl do you need to make 450ml of a 0.5M Tris, 500mM KCl 10x stock solution? (MW tris = 121.1g/mole, MW KCl =74.6g/mole) [round to the nearest tenths place, record your answer as a number (gram units are supplies in the question)]
Using the stock solution from the previous question, what is the mM concentration of the KCl in the working solution? [record your answer as a number only (mM units are supplied in the question)]
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