BIOCHEMISTRY Please help with what you can Will give high ra

Name: Part Use the following protein sequence to answer the following questions (20 pts total) mhgnnghsvp pskrsetrap vapagcnggy paeawnfwtr kglvagglye shvgcrpysi 61 ppcehhvngs rppctgegdt p kcskicepg ysptykqdkh ygynsysvsn sekdimaeiy 12 kngpvegaf vysdfllyks gvyghvtgem mgghairilg wgvengtpyw lvanswntdW 181 gdngffkilr gqdhcgiese vvagiprtdq yweki 1. Take the following sequence and do a blast search (h nlm nih.gov/B and select protein blast on the right, paste the sequence in its entirety in white box and scroll to the bottom of the webpage and click blast\") and determine (5 pts) a. What protein is encoded by this protein? b. What superfamily does this protein belong to? c. What organism is this protein found in? homo saniens d. Which cellular organelle the protein is located in. NSOSOMes e. What metabolic role does this enzyme have? ahanD3 AMVHN mdfase, 2. You decide to purify the protein for enzymatic studies. Based on what you know about the protein (info listed in question 1), which of the following buffers would you choose to use for purification. Explain why. (3 pts) Buffer Citric Acid, mono rate 4.76 MES 6.15 PIPES 6.8 8.2 Trizma 3. Show calculations for a quick approximation ofthe molecular weight of the protein above. (2 pts) 4. Using the following website (ht expas.orglcompute paste sequence in white box and click \"click here to compute button. a. What is the pl? (2 pts) b. what is the molecular weight? (2 pts) 5. Based on the pl, at pH 5 a. What would be the expected charge? (3 pts) b. the best type of column to use for ion exchange chromatography? Explain. (3 What would be pts)

Solution

Answer 1.

Cathepsin B

Cathepsin B belongs to the superfamily of papain-like cysteine proteases

Humans Homo Sapiens

Cathepsin B asts as a lysosomal cysteine protease in normal cells and tissues

cathepsin B is involved in the turnover of proteins and plays various roles in maintaining the normal metabolism of cells

Answer 2

Assay for cathepsin B activity: In a 500 pL quartz cuvette at 25 \"C, IO pL of enzyme sample was combined with 480 pL of buffer (88 mM KH2P04, 12 mM Na2HP04, 1.33 mM EDTA, 2.7 mM DTT, 0.03% Brij 35, pH 5.8) and 10 pL of the substrate stock solution of Z-Arg-Arg-AMC (Peptides International, Louisville, Kentucky; 80 mM in DMSO). Excitation at 380 nm and emission at 440 nm were monitored on a LS5OB Luminescence Spectrometer (Perkin Elmer) for activity determinations. For activation of baculovirus-derived samples prior to purification, these samples were applied to PD-IO columns (Pharmacia, Piscataway, New Jersey) pre-equilibrated with 50 mM sodium acetate, pH 5.0 with 1 mM EDTA, and were eluted in 3.5 mL of the same buffer. Samples were stored overnight at 4°C to allow full activation in this buffer. This assay was used to monitor purification of the enzyme and to study the kinetic characteristics of the recombinant products. Cathepsin B was activated by dialysis to SP-Sepharose Buffer (pH 5.0) and incubation at 4 \'C overnight. For kinetic analyses, the amount of active enzyme was determined by titration with the thiol protease inhibitor E64.

Answer 3.

Molecular weight is calculated by techniques ELISA, WB, IHC, IF, FC

A375 cells were subjected to SDS PAGE followed by western blot with 12216-1-AP(CTSB antibody) at dilution of 1:800

Immunohistochemistry of paraffin-embedded human ovary tumor tissue slide using 12216-1-AP( Cathepsin B Antibody) at dilution of 1:200 (under 10x lens).

Immunohistochemistry of paraffin-embedded human ovary tumor tissue slide using 12216-1-AP( Cathepsin B Antibody) at dilution of 1:200 (under 40x lens).

Immunofluorescent analysis of A375 cells using 12216-1-AP( Cathepsin B Antibody) at dilution of 1:25 and Alexa Fluor 488-congugated AffiniPure Goat Anti-Rabbit IgG(H+L)

1X10^6 MCF-7 cells were stained with 0.2ug Cathepsin B antibody (12216-1-AP, red) and control antibody (blue). Fixed with 90% MeOH blocked with 3% BSA (30 min). Alexa Fluor 488-congugated AffiniPure Goat Anti-Rabbit IgG(H+L) with dilution 1:1500.

Species specificity:

Human, Mouse, frog

Positive WB detected in:

A375 cells, HT-1080 cells, human brain tissue, human liver tissue, mouse brain tissue, NIH/3T3 cells, RAW264.7 cells

Positive IHC detected in:

human ovary tumor tissue, human breast cancer tissue, human colon cancer tissue

Positive IF detected in:

A375 cells

Positive FC detected in:

MCF-7 cells

Recommended dilution:

WB : 1:200-1:2000

IHC : 1:100-1:400

IF : 1:10-1:100

Product Information

Source:

Rabbit

Purification method:

Antigen affinity purification

Isotype:

IgG

Storage:

PBS with 0.1% sodium azide and 50% glycerol pH 7.3. Store at -20^oC. Aliquoting is unnecessary for -20^oC storage.

Immunogen Information

Immunogen:

Cathepsin B fusion protein ag2898

Full name:

cathepsin B

Calculated molecular weight:

229aa,38 kDa

Observed molecular weight:

38 kDa, 44 kDa

GenBank accession number:

BC010240

Gene ID (NCBI):

1508

Gene symbol

CTSB

Synonyms

APP secretase, APPS, cathepsin B, Cathepsin B1, CPSB, CTSB