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92516
ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit
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ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit #92516

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Confocal immunofluorescent analysis of fixed frozen hippocampus from C57BL/6 (left) and APOE4 knock-in (hu/hu, model #1549-F, right) mice labeled with ApoE (pan) (D7I9N) Rabbit mAb (top, green). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to colabeling with GFAP (GA5) Mouse mAb (Alexa Fluor® 594 Conjugate) #8152 (bottom, red), and AQP4 (D1F8E) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #89851 (bottom, blue pseudocolor). Mice from Taconic Biosciences, Inc.
Simple Western™ analysis of lysates (0.1 mg/mL) from mouse brain using PSD95 (D27E11) XP® Rabbit mAb #3450. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
Simple WesternTM analysis of lysates (0.75 mg/mL) from Mouse Brain cells using Phospho-PSD95 (Ser295) (A8F8Z) Rabbit mAb #45737. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
CUT&RUN was performed with 293 cells treated with Forskolin #3828 (30 μM) for 1h and either CREB (48H2) Rabbit mAb or Phospho-CREB (Ser133) (87G3) Rabbit mAb #9198, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across NR4A3 gene.
Simple Western™ analysis of lysates (0.1 mg/mL) from SK-N-MC cells treated with Forskolin (30uM, 30min) and IBMX (0.5mM, 30 min) using Phospho-CREB (Ser133) (87G3) Rabbit mAb #9198. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. This antibody also detects the phosphorylated form of the CREB-related protein, ATF-1 (as indicated). The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western blot analysis of extracts from mouse brain, rat brain, and rat prefrontal cortex tissues using AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb.
Western blot analysis of cell extracts from Hep G2 cells treated with Brefeldin A #9972 (10 ng/ml, 90 min) and human cerebellum using ApoE (pan) (D7I9N) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human kidney using ApoE (pan) (D7I9N) Rabbit mAb.
Western blot analysis of extracts from various cell lines and tissues using LRP1 (E2Q7S) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Western blot analysis of extracts from human cerebellum and rat brain using PSD95 (D27E11) XP® Rabbit mAb.
Western blot analysis of extracts from mouse brain, untreated (-) or λ phosphatase-treated (+), using Phospho-PSD95 (Ser295) (A8F8Z) Rabbit mAb (upper) or PSD95 (D74D3) XP® Rabbit mAb #3409 (lower).
Western blot analysis of extracts from CAD cells and neonatal mouse brain using Synapsin-1 (D12G5) XP® Rabbit mAb.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western Blot analysis of extracts from SK-N-MC, COS, NIH/3T3, C6 and Drosophila S2 cells, using CREB (48H2) Rabbit mAb.
Western blot analysis of extracts from SK-N-MC cells, untreated or forskolin- and FGF-treated, using Phospho-CREB (Ser133) (87G3) Rabbit mAb (upper) or CREB (48H2) Rabbit mAb #9197 (lower).
Western blot analysis of cell extracts from 293T cells, treated with Brefeldin A #9972 (10 ng/ml, 90 min; +) and mock transfected (-) or transfected with a construct expressing full-length human ApoE2 (hApoE2; +), ApoE3 (hApoE3; +), or ApoE4 (hApoE4; +), using ApoE (pan) (D7I9N) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Immunohistochemical analysis of paraffin-embedded liver (left) or brain (right) from C57BL/6NTac (wt/wt, model #B6-F, top) and APOE4 knock-in (hu/hu, model #1549-F, bottom) mice using ApoE (pan) (D7I9N) Rabbit mAb. Mice from Taconic Biosciences, Inc.
Confocal immunofluorescent analysis of MEF-1 cells (left, LRP1 (+/+), positive) or PEA 13 cells (right, LRP1 (-/-), negative) using LRP1 (E2Q7S) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Confocal immunofluorescent analysis of rat cerebellum and retina using PSD95 (D27E11) XP® Rabbit mAb (red) and Neurofilament-L (DA2) Mouse mAb #2835 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunohistochemical analysis of paraffin-embedded human cerebellum using Synapsin-1 (D12G5) XP® Rabbit mAb.
Western blot analysis of extracts from mouse brain, untreated (-) or λ-phosphatase-treated (+), using Phospho-AMPA Receptor 1 (GluA1) (Ser831) (A5O2P) Rabbit mAb (upper) and AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb #13185 (lower).
Immunoprecipitation of CREB from SK-N-MC extracts. Lane 1 is CREB (48H2) Rabbit mAb, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is 10% input. Western blot analysis was perfomed using CREB (86B10) Mouse mAb #9104. Anti-mouse IgG, HRP-linked Antibody #7076 was used as a secondary antibody.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, showing nuclear staining, using Phospho-CREB (Ser133) (87G3) Rabbit mAb.
Immunoprecipitation of AMPA Receptor 1 (GluA1) from mouse brain extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb.
Immunoprecipitation of ApoE from Hep G2 cells treated with Brefeldin A #9972 (10 ng/ml, 90 min), using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or ApoE (pan) (D7I9N) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using ApoE (pan) (D7I9N) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human spleen using ApoE (pan) (D7I9N) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse cortex using Synapsin-1 (D12G5) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human astrocytoma, using CREB (48H2) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse lung using Phospho-CREB (Ser133) (87G3) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human small intestine using ApoE (pan) (D7I9N) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded rat retina using Synapsin-1 (D12G5) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using CREB (48H2) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-CREB (Ser133) (87G3) Rabbit mAb in the presence of control peptide (left) or Phospho-CREB (Ser133) Blocking Peptide #1090 (right).
Confocal immunofluorescent analysis of mouse hippocampus using AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunohistochemical analysis of paraffin-embedded human skin using ApoE (pan) (D7I9N) Rabbit mAb in the presence of control peptide (left) and antigen-specific peptide (right).
Confocal immunofluorescent analysis of mouse brain using Synapsin-1 (D12G5) XP® Rabbit mAb (green) and β3-Tubulin (TU-20) Mouse mAb #4466 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunohistochemical analysis of paraffin-embedded human Non-Hodgkin's lymphoma, using CREB (48H2) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded SK-N-MC cells, untreated (left) or IBMX- and forskolin-treated (right), showing induced nuclear staining, using Phospho-CREB (Ser133) (87G3) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded 293T cell pellets, control (left-top) or transfected with human ApoE isoforms ApoE2 (right-top), ApoE3 (left-bottom), and ApoE4 (right-bottom) using ApoE (pan) (D7I9N) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse brain, using CREB (48H2) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human renal cell carcinoma, untreated (left) or lambda phosphatase-treated (right), using Phospho-CREB (Ser133) (87G3) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded Hep G2 cell pellet (left, positive) or 293T cell pellet (right, negative) using ApoE (pan) (D7I9N) Rabbit mAb.
Confocal immunofluorescent analysis of mouse cerebellum labeled with CREB (48H2) Rabbit mAb (red) and Neurofilament-L (DA2) Mouse mAb #2835 (green). Blue pseudocolor =DRAQ5® #4084 (fluorescent DNA dye).
Confocal immunofluorescent images of rat dentate gyrus, either sham-operated (left) or 15 min ischemia followed by 30 min (center) and 4 h (right) reperfusion, labeled with Phospho-CREB (Ser133) (87G3) Rabbit mAb (red), Neurofilament-L (DA2) Mouse mAb #2835 (blue) and Phospho-S6 Ribosomal Protein (Ser235/236) (2F9) Rabbit mAb (Alexa Fluor® 488 Conjugate) #4854.
Confocal immunofluorescent analysis of Hep G2 cells (left, positive) and 293T cells (right, negative) using ApoE (pan) (D7I9N) Rabbit mAb #13366 (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Confocal immunofluorescent analysis of SK-N-MC cells showing nuclear stain with CREB (48H2) Rabbit mAb (A, red) compared to an isotype control (B). Blue pseudocolor =DRAQ5® (fluorescent DNA dye).
Confocal microscopic images of SK-N-MC cells showing nuclear stain after 25 minute treatment with Forskolin and IBMX using Phospho-CREB (Ser133) (87G3) Rabbit mAb (left, red) compared to untreated cells (right). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of Jurkat cells using CREB (48H2) Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with TPA #4174 (500nM) and Ionomycin #9995 (1uM,4hrs;green) using Phospho-CREB (Ser133) (87G3) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells treated with Forskolin #3828 (30 μM) for 1h and either Phospho-CREB (Ser133) (87G3) Rabbit mAb or CREB (48H2) Rabbit mAb (#9197), using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across NR4A3, a known target gene of both Phospho-CREB and CREB (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells, treated with Forskolin #3828 (30 μM) for 1h and either 10 μl of CREB (48H2) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human ALS2 exon 1 primers, SimpleChIP® Human NR4A3 Promoter Primers #4829, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells treated with Forskolin #3828 (30 μM) for 1h and either Phospho-CREB (Ser133) (87G3) Rabbit mAb or CREB (48H2) Rabbit mAb (#9197), using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 9 (upper), including NR4A3 (lower), a known target gene of both Phospho-CREB and CREB (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells treated with Forskolin #3828 (30 μM) for 1h and either Phospho-CREB (Ser133) (87G3) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human ALS2 exon 1 primers, SimpleChIP® Human NR4A3 Promoter Primers #4829, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Western blot analysis of extracts from HepG2 cells (lane 1), 293T mock transfected (lane 2) or transiently transfected with a construct expressing ApoE4 (lane 3), whole liver extracts from wild type C57BL/6NTac model #B6-F mice (lane 4), or ApoE4 knock-in (model #1549-F) (lane 5), whole brain extracts from wild type C57BL/6NTac mice (lane 6), or ApoE4 knock-in (lane 7) using ApoE (pan) (D7I9N) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). Mice from Taconic Biosciences, Inc.
CUT&RUN was performed with 293 cells treated with Forskolin #3828 (30 μM) for 1h and Phospho-CREB (Ser133) (87G3) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across NR4A3 gene.
CUT&RUN was performed with 293 cells treated with Forskolin #3828 (30 μM) for 1h and Phospho-CREB (Ser133) (87G3) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-Seq, CUT&RUN) #56795. The figures show binding across chromosome 9 (upper), including NR4A3 (lower) gene.
CUT&RUN was performed with 293 cells treated with Forskolin #3828 (30 μM) for 1h and either Phospho-CREB (Ser133) (87G3) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human ALS2 exon 1 primers and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
To Purchase # 92516T
Cat. # Size Price Inventory
92516T
1 Kit  (9 x 20 microliters)

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
LRP1 (E2Q7S) Rabbit mAb 26387 20 µl
  • WB
  • IF
H M R 85 Rabbit IgG
ApoE (pan) (D7I9N) Rabbit mAb 13366 20 µl
  • WB
  • IP
  • IHC
  • IF
H 35 Rabbit IgG
PSD95 (D27E11) XP® Rabbit mAb 3450 20 µl
  • WB
  • IF
H M R 95 Rabbit IgG
Phospho-PSD95 (Ser295) (A8F8Z) Rabbit mAb 45737 20 µl
  • WB
H M R 95 Rabbit IgG
Synapsin-1 (D12G5) XP® Rabbit mAb 5297 20 µl
  • WB
  • IP
  • IHC
  • IF
H M R 77 Rabbit IgG
AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb 13185 20 µl
  • WB
  • IP
  • IF
M R 100 Rabbit IgG
Phospho-AMPA Receptor 1 (GluA1) (Ser831) (A5O2P) Rabbit mAb 75574 20 µl
  • WB
  • IP
H M 100 Rabbit IgG
CREB (48H2) Rabbit mAb 9197 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
  • C&R
H M R Mk Dm 43 Rabbit IgG
Phospho-CREB (Ser133) (87G3) Rabbit mAb 9198 20 µl
  • WB
  • IHC
  • IF
  • F
  • ChIP
  • C&R
H M R 43 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit provides an economical means of detecting key synaptic signaling pathways in response to ApoE-mediated LRP1 activation by western blot. The kit includes enough antibodies to perform at least two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each total antibody in the ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit detects endogenous levels of its target protein. LRP1 (E2Q7S) Rabbit mAb detects endogenous levels of the β subunit of LRP1. ApoE (pan) (D7I9N) Rabbit mAb also recognizes overexpressed ApoE2, ApoE3, and ApoE4 proteins. CREB (48H2) Rabbit mAb does not cross-react with other ATF/CREB family members. The antigen for Synapsin-1 (D12G5) XP® Rabbit mAb is 100% conserved between human synapsin-1a and synapsin-1b. Each phospho-specific antibody in the ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit detects endogenous levels of CREB only when phosphorylated at Ser133, PSD95 protein only when phosphorylated at Ser295, and AMPA Receptor 1 (GluA1) protein only when phosphorylated at Ser831. Phospho-CREB (Ser133) (87G3) Rabbit mAb also detects the phosphorylated form of the CREB-related protein, ATF-1. While Phospho-AMPA Receptor 1 (GluA1) (Ser831) (A5O2P) Rabbit mAb refers to Ser831, consistent with the literature, it is Ser849 in the UniProt sequence P42261.

Source / Purification

Monoclonal antibodies to total proteins are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu4488 of human LRP1 protein, Pro285 of human ApoE protein,Gln53 of human PSD95 protein, Gln483 of human synapsin-1 protein, Ala275 of human AMPA Receptor 1 (GluA1) protein, and recombinant protein specific to the amino terminus of human CREB-1 protein. Phospho-specific monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser295 of human PSD95 protein, Ser831 of human AMPA Receptor 1 (GluA1) protein, and Ser133 of human CREB protein.

Background

Low density lipoprotein receptor related protein 1 (LRP1) is a type I transmembrane receptor that mediates the endocytosis of various ligands, including apolipoproteins and tau. Both proteins are genetically and pathologically linked to Alzheimer’s disease (AD) (1,2). Human apolipoprotein E (ApoE) is a component of circulating lipoproteins when three human genetic ApoE variants, ApoE2, ApoE3, and ApoE4, exhibit distinct receptor-binding properties and differentially contribute to AD progression through a cellular mechanism that is poorly understood (2). Altered synaptic signaling is one proposed mechanism that contributes to altered neuronal function, which correlates with disease (3-5). Postsynaptic Density protein 95 (PSD95) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that functions as a scaffolding protein to promote assembly and function of the postsynaptic density complex (6,7). At the presynapse, synapsins function to regulate neurotransmitter release (8,9). Dynamic phosphorylation of PSD95 at Ser295 reflects synaptic signaling that may alter synaptic function (10). In addition to PSD95, postsynaptic glutamate receptors, including AMPA-(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, can be directly phosphorylated. Phosphorylation of AMPA Receptor 1 (GluA1) at either Ser831 or Ser845 alters AMPA receptor ion channel function to change synaptic efficacy (11). CREB is a bZIP transcription factor that activates target genes through cAMP response elements. CREB is activated by phosphorylation at Ser133 by various signaling pathways including Erk, Ca2+,stress signaling, as well as synaptic signaling (3). 

Pathways

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Limited Uses

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For Research Use Only. Not for Use in Diagnostic Procedures.
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