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AB257594

Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell lysate

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PNP KO cell lysate available now. KO validated by Western blot. Free of charge wild type control included. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 2.

View Alternative Names

Purine nucleoside phosphorylase, Inosine phosphorylase, NP, Inosine-guanosine phosphorylase, PNP

3 Images
Western blot - Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell lysate (AB257594)
  • WB

Lab

Western blot - Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell lysate (AB257594)

Lane 1 : Wild-type HeLa cell lysate (20 ug)
Lane 2 : PNP knockout HeLa cell lysate (20 ug)
Lane 3 : Jurkat cell lysate (20 ug)
Lane 4 : JAR cell lysate (20 ug)

ab109559 was shown to specifically react with Nucleoside phosphorylase in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab266158 (knockout cell lysate ab257594) was used. Wild-type and Nucleoside phosphorylase knockout samples were subjected to SDS-PAGE. ab109559 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Nucleoside phosphorylase antibody [EPR5714] (<a href='/en-us/products/primary-antibodies/nucleoside-phosphorylase-antibody-epr5714-ab109559'>ab109559</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PNP knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-pnp-nucleoside-phosphorylase-knockout-hek-293t-cell-line-ab266158'>ab266158</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

JAR cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 32 kDa

Observed band size: 31 kDa

false

Western blot - Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell lysate (AB257594)
  • WB

Lab

Western blot - Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell lysate (AB257594)

Lane 1 : Wild-type HeLa cell lysate (20 ug)
Lane 2 : PNP knockout HeLa cell lysate (20 ug)
Lane 3 : Jurkat cell lysate (20 ug)

ab109447 was shown to specifically react with Nucleoside phosphorylase in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab266158 (knockout cell lysate ab257594) was used. Wild-type and Nucleoside phosphorylase knockout samples were subjected to SDS-PAGE. ab109447 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Nucleoside phosphorylase antibody [EPR5715] (<a href='/en-us/products/primary-antibodies/nucleoside-phosphorylase-antibody-epr5715-ab109447'>ab109447</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PNP knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-pnp-nucleoside-phosphorylase-knockout-hek-293t-cell-line-ab266158'>ab266158</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 32 kDa

Observed band size: 31 kDa

false

Sanger Sequencing - Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell lysate (AB257594)
  • Sanger seq

Unknown

Sanger Sequencing - Human PNP (Nucleoside phosphorylase) knockout HEK-293T cell lysate (AB257594)

Homozygous : 1 bp insertion in exon 2

Key facts

Cell type

HEK-293T

Species or organism

Human

Tissue

Kidney

Knockout validation

Sanger Sequencing,Western blot

Mutation description

Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 2.

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Reactivity data

{ "title": "Reactivity Data", "filters": { "stats": ["", "Reactivity", "Dilution Info", "Notes"] }, "values": { "WB": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" } } }
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Product details

Knockout cell lysate achieved by CRISPR/Cas9.

REACH authorisation
Abcam has not and does not intend to apply for the REACH Authorisation of customers' uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version. Please refer to our lysis protocol for further details on how our lysates are prepared.

User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.

This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.

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What's included?

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Properties and storage information

Gene name
PNP
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Zygosity
Homozygous
Shipped at conditions
Ambient - Can Ship with Ice
Appropriate short-term storage conditions
-20°C
Appropriate long-term storage conditions
-20°C
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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

Nucleoside phosphorylase also known as PNP or purine nucleoside phosphorylase is an enzyme with a vital mechanical role in nucleoside metabolism. The enzyme facilitates the phosphorolytic cleavage of the glycosidic bond in nucleosides producing ribose 1-phosphate and free bases such as guanine and hypoxanthine. This catalytic function plays an important part in nucleotide salvage pathways. PNP has a molecular mass of approximately 32000 Daltons and shows expression predominantly in the lymphoid tissues including the thymus and spleen.
Biological function summary

Nucleoside phosphorylase is important in maintaining nucleotide homeostasis. It operates as a monomer or part of a homotrimeric complex which allows it to efficiently catalyze its reactions in purine metabolism. The absence or dysfunction of PNP results in the accumulation of nucleosides and diminished levels of nucleotide pools which can severely hamper DNA replication and repair.

Pathways

Nucleoside phosphorylase plays an integral role in the purine salvage pathway which is vital for recycling purines to form new nucleotides. It works alongside other enzymes such as adenine phosphoribosyltransferase to conserve energy by recycling purines. This pathway connects closely with the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) pathway showing how PNP is intertwined in broader nucleotide biosynthesis and degradation processes.

Deficiencies in nucleoside phosphorylase correlate strongly with immunodeficiencies notably purine nucleoside phosphorylase deficiency (PNP deficiency) which leads to compromised T-cell immunity. This condition can result in recurrent infections and developmental delay. Additionally the enzyme's dysregulation associates with certain leukemias where altered nucleotide pools contribute to the proliferation of malignant cells. In these contexts PNP interacts with proteins central to these disorders including those involved in purine metabolism placing it at a critical junction for potential therapeutic intervention.
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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 2 US: 2

Adherent/suspension

Adherent

Gender

Female

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Product protocols

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Product promise

We are committed to supporting your work with high-quality reagents, and we're here for you every step of the way. In the unlikely event that one of our products does not perform as expected, you're protected by our Product Promise.
For full details, please see our Terms & Conditions

Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.

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