My colleague and I have been working on developing an ELISA for various analytes. We are accounting for matrix effects so we spike our recombinant protein into a matrix and it works fine. When we try and validate our internal standard by doing a 2-fold serial diltution the target analyte seems to be inactivated. That is the undiluted sample works fine but when we do the serial dilution in a matrix there is no signal. We have tried several different matrices: bovine, sheep, goat. We have heat-inactivated and added protease inhibitors to the bovine matrix with no positve results. If any one out there has any ideas, or solutions as to why this might be happening it would be greatly appreciated.

Thank you in advance for your help

1. DNA ligase links two _______ DNA fragments by ______bonds.
a. complementary; hydrogen
b. circular, covalent
c. palindromic, covalent
d. linear, covalent
e. linear, hydrogen.

2. One feature of "engineered" plasmids that is helpful in the isolation and analysis of cloned DNA is:
a. they can handle DNA fragments up to 10 kb.
b. that they are an integral part of all eukaryotic cells
c. they contain no genetic material of their own so that the cloned fragment is truly isolated.
d. the presence of genes that allow transformed cells to survive on different media.
e. all of the above.

3. During the preparation of a human genomic library, plasmids containing human DNA fragments are inserted into:
a. compatible human cells.
b. antibiotic resistant strain of E. colo to protect the inserted fragments.
c. antibiotic sensitive E. Coil cells that become antibiotic resistant if transformed.
d. bacteriophage’s.
e. mRNA molecules

4. If the amino acid sequence of a protein is known, then one can use the sequence to synthesize a ____ that will hybridize with the DNA of the gene that produces the protein.
a. plasmid
b. recombinant protein
c. antibody
d. genetic probe
e. bacteriophage.

Thank you so much!

Heparin, a drug used to prevent clotting post-operatively, saves many lives and is a recombinant protein made from cells from pigs, there is a cow version but it doesn’t work as well and leads to deadly HIT complications. Would a muslim use this product or would he risk death for his beliefs? What about other life-saving porcine medications?

A few months ago, someone asked the CDC an embarrassing question concerning dogs and HIV antibodies:

"Dear CDC, …

Why are half of dogs ‘HIV’-positive, and if my breeder dog Cerberus, whom I love deeply turns out to be positive (he tested positive, then negative, then positive, then negative again), should I have him put to sleep?

EG. (Cancer Res 1990 Sep 1;50(17 Suppl):5628S-5630S Studies with canine sera that contain antibodies which recognize human immunodeficiency virus structural proteins. Strandstrom HV, Higgins JR, Mossie K, Theilen GH. College of Veterinary Medicine, Helsinki, Finland):

Abstract. In a serological survey, using the immunoblotting technique, we found that substantial numbers of dog sera from both normal and diseased dogs, including dogs with neoplasia, reacted with one or more human immunodeficiency virus (HIV) recombinant proteins. A total of 144 dog sera were tested, and 72 (50%) of them reacted with one or more HIV recombinant structural proteins. Ten dog sera were also tested for reactivity with simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and caprine arthritis encephalitis virus (CAEV). Six dog sera reacted with at least the major core protein of HIV, while one of the dog sera tested reacted with SIV core protein, and there were no reactions with the viral proteins of either FIV or CAEV. Cell extracts from canine peripheral blood lymphocytes cocultivated with human cells and an extract of human cells infected with HIV were immunoblotted against dog sera which previously tested positive or negative on HIV recombinant protein commercially available Western blot strips. Two lymphocyte lysates and the HIV-infected Hut cell lysate reacted with the Western blot strip-positive dog serum; however, no reactions were seen with the Western blot strip-negative dog serum)."

Incidently, it has also been demonstrated that goats and cows are also known to test positive using the current "HIV" test kits, EG. (Willman et al., Heterophile Antibodies to Bovine and Caprine Proteins Causing False-Positive Human Immunodeficiency Virus Type 1 and Other Enzyme-Linked Immunosorbent Assay Results. Clinical and Diagnostic Laboratory Immunology, p. 615-616, Vol. 6, No. 4, July 1999)

Remarkably, the CDC responded soon afterward:

"Thank you for writing CDC STD/HIVNet.

Dogs do NOT become infected with the Human Immunodeficiency Virus (HIV). A dog would not test positive for HIV. HIV is the Human Immunodeficiency Virus. HIV is a member of the human retrovirus family. All viruses and retroviruses are simple microbes that have no metabolism and cannot function independently of other life forms. They lack the basic machinery for reproduction and must invade other living organisms to reproduce or replicate. In other words, retroviruses and viruses rely on the cells of the host for reproduction to survive. Each retrovirus has a specific host. For example, a human retrovirus, like HIV, requires a human host. So a human retrovirus cannot survive in other animals or insects. Animals, like dogs, have their own retroviruses. These animal
retroviruses do not affect humans.

The article (Standtrom et al.) seems to be stating that antibodies (substances produced by the body to fight infection) in a dog’s blood reacted to structural proteins of the virus (HIV) and NOT stating that dogs are infected with HIV."

This answer from the CDC prompted the following thoughts:

"It seems to me that the really important point to make from this is not just that dogs, cows, sheep, have ‘HIV surrogate markers’ but never develop ‘AIDS’. The more important question I take from the studies you mentioned would be: Why, when antibodies of a dog react to certain proteins manufactured in a lab, is this interpreted as ‘antibodies reacting to structural proteins of HIV’ but at the same time NOT evidence that ‘[these] dogs are infected with HIV’, yet when antibodies of a human react to exactly the same proteins, this is taken as evidence of ‘HIV’ infection?

The CDC’s interpretation of Strandstrom et al seems to be an (indirect) admission that simply detecting a certain combination of antibodies to proteins in blood is not sufficient to conclude infection with an exogenous retrovirus. Certainly, they believe felines are capable of such retroviral infection, as I see countless webpages devoted to ‘FIV’ (’feline immunodeficiency virus’). So, my question to the CDC would be: how do they know that detection of a certain combination of antibodies to proteins indicates infection with an exogenous retrovirus in a human, but not in a dog? An equally reasonable interpretation might be that these combinations of antibodies indicate infection with an exogenous retrovirus in a dog, but not a human. So, how do they know it is one
case and not the other?

The only way I can see the CDC can claim infection in one case and not the other, is if some additional validation process had been achieved in

Why are half of dogs ‘HIV’-positive, and if my breeder dog Cerberus, whom I love deeply turns out to be positive (he tested positive, then negative, then positive, then negative again), should I have him put to sleep?

Studies with canine sera that contain antibodies which recognize human immunodeficiency virus structural proteins. Strandstrom HV, Higgins JR, Mossie K, Theilen GH. College of Veterinary Medicine, Helsinki, Finland):

Abstract. In a serological survey, using the immunoblotting technique, we found that substantial numbers of dog sera from both normal and diseased dogs, including dogs with neoplasia, reacted with one or more human immunodeficiency virus (HIV) recombinant proteins. A total of 144 dog sera were tested, and 72 (50%) of them reacted with one or more HIV recombinant structural proteins. Ten dog sera were also tested for reactivity with simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and caprine arthritis encephalitis virus (CAEV). Six dog sera reacted with at least the major core protein of HIV, while one of the dog sera tested reacted with SIV core protein, and there were no reactions with the viral proteins of either FIV or CAEV. Cell extracts from canine peripheral blood lymphocytes cocultivated with human cells and an extract of human cells infected with HIV were immunoblotted against dog sera which previously tested positive or negative on HIV recombinant protein commercially available Western blot strips. Two lymphocyte lysates and the HIV-infected Hut cell lysate reacted with the Western blot strip-positive dog serum; however, no reactions were seen with the Western blot strip-negative dog serum)."

Incidently, it has also been demonstrated that goats and cows are also known to test positive using the current "HIV" test kits, EG. (Willman et al., Heterophile Antibodies to Bovine and Caprine Proteins Causing False-Positive Human Immunodeficiency Virus Type 1 and Other Enzyme-Linked Immunosorbent Assay Results. Clinical and Diagnostic Laboratory Immunology, p. 615-616, Vol. 6, No. 4, July 1999)
Remarkably, the CDC responded soon afterward:

Dogs do NOT become infected with the Human Immunodeficiency Virus (HIV). A dog would not test positive for HIV. HIV is the Human Immunodeficiency Virus. HIV is a member of the human retrovirus family. All viruses and retroviruses are simple microbes that have no metabolism and cannot function independently of other life forms. They lack the basic machinery for reproduction and must invade other living organisms to reproduce or replicate. In other words, retroviruses and viruses rely on the cells of the host for reproduction to survive. Each retrovirus has a specific host. For example, a human retrovirus, like HIV, requires a human host. So a human retrovirus cannot survive in other animals or insects. Animals, like dogs, have their own retroviruses. These animal
retroviruses do not affect humans.
The article (Standtrom et al.) seems to be stating that antibodies (substances produced by the body to fight infection) in a dog’s blood reacted to structural proteins of the virus (HIV) and NOT stating that dogs are infected with HIV."

This answer from the CDC prompted the following thoughts:
It seems to me that the really important point to make from this is not just that dogs, cows, sheep, have ‘HIV surrogate markers’ but never develop ‘AIDS’. The more important question I take from the studies you mentioned would be: Why, when antibodies of a dog react to certain proteins manufactured in a lab, is this interpreted as ‘antibodies reacting to structural proteins of HIV’ but at the same time NOT evidence that ‘[these] dogs are infected with HIV’, yet when antibodies of a human react to exactly the same proteins, this is taken as evidence of ‘HIV’ infection?

The CDC’s interpretation of Strandstrom et al seems to be an (indirect) admission that simply detecting a certain combination of antibodies to proteins in blood is not sufficient to conclude infection with an exogenous retrovirus. Certainly, they believe felines are capable of such retroviral infection, as I see countless webpages devoted to ‘FIV’ (’feline immunodeficiency virus’). So, my question to the CDC would be: how do they know that detection of a certain combination of antibodies to proteins indicates infection with an exogenous retrovirus in a human, but not in a dog? An equally reasonable interpretation might be that these combinations of antibodies indicate infection with an exogenous retrovirus in a dog, but not a human. So, how do they know it is onease and not the other?
The only way I can see the CDC can claim infection in one case and not the other, is if some additional validation process had been achieved in the one case and failed in the other. I’m not aware of any such process, as all such ‘validation processes’ that I’m aware of consist of simply testing WB against itself (reproducibility) or using some vague combination of indi

If the amino acid sequence of a protein is known, then one can use the sequence to synthesize a ____________ that will hybridize with the DNA of the gene that produces the protein.

A. plasmid

B. recombinant protein

C. antibody

D. genetic probe

E. bacteriophage

"what type of purification did you see with the recombinant protein?".
what is this question asking? types of chromatography used, like Ni affinity chromatography???
how could i answer this question?

I am expressing a recombinant protein in E coli with a his tag. In order to remove this tag, I am digesting it with thrombin. After digestion I get both mine recombinant protein and thrombin. How can I get ride off thrombin?

I guess I just want a couple of people to confirm this. From what i’ve read to make a recombinant protein (that is artificially produce a protein) you mircoinject a bacterium with the sequence of dna that produces that protein. The bacteria you injected the sequence with will multiply and produce said protein correct?