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Resources results for "LAIR-1"

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Our Calpain 1 Large Subunit (Mu-type) Antibody #2558 detects endogenous levels of total calpain 1 (large subunit) protein. The antibody detects full-length calpain 1 as well as calpain 1 autoproteolytically cleaved at leucine 28. The intensity of the cleaved band can vary depending on the species and type of cell lysate used.

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We do not perform enzymatic activity assays internally using the Cell Lysis Buffer (10X) #9803, so we are unable to guarantee that the enzymatic activity will be conserved. We routinely use this buffer in immunoassays such as western immunoblot, IP, and ELISA without issue.

As a reference, the components of the Cell Lysis Buffer (10X) #9803 are provided below:
20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin

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When we validate an antibody for Simple Western™ we titrate the antibody dilution against a broad range of lysate concentrations. We recommend testing at least three antibody dilutions (1:10, 1:50 and 1:250) across a range of lysate concentrations (0.1 mg/mL – 1 mg/mL).

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We typically recommend the PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 that comes with the Glucose-6-Phosphate Dehydrogenase (G6PD) Activity Assay Kit #12581 as it has been validated for use with the kit. The PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 was made specifically for this assay kit and for ELISA formats. We have not tested any other lysis buffers with the Glucose-6-Phosphate Dehydrogenase (G6PD) Activity Assay Kit #12581, so we cannot guarantee the performance. The ingredients of the PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 are as follows: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% Triton, 20 mM sodium Pyrophosphate, 25 mM Sodium Fluoride, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 ug/ml leupeptin.

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The regions recognized by our PD-1 and PD-L1 antibodies are as follows:

PD-1 (EH33) Mouse mAb (IHC-Specific) #43248 binds the extracellular domain.
PD-1 (D7D5W) XP® Rabbit mAb (Mouse Specific) #84651 binds the intracellular domain.
PD-1 (D4W2J) XP® Rabbit mAb #86163 binds the intracellular domain.
PD-L1 (E1L3N®) XP® Rabbit mAb #13684 binds the intracellular domain.
PD-L1 (Extracellular Domain Specific) (E1J2J) Rabbit mAb #15165 binds the extracellular domain.
PD-L1 (405.9A11) Mouse mAb #29122 binds the intracellular domain.
PD-L1 (D4H1Z) Rabbit mAb #60475 binds the extracellular domain.
PD-L1 (D5V3B) Rabbit mAb (Mouse Specific; IHC Specific) #64988 binds the intracellular domain.
PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb #86744 binds the extracellular domain.

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CIP/Lambda Phosphatase Treatment of Cell Lysates:

1) Wash cells 2X with ice-cold PBS.

2) Add cold 1X cell lysis buffer that does not include phosphatase inhibitors [1-2 E7 cells per ml or 0.4ml per 10 cm plate (80-90% confluent)]. Our cell lysis buffer is prepared as follows:

20 mM Tris-HCl (pH 7.5)
150 mM NaCl
1 mM Na2EDTA
1 mM EGTA
1% Triton
1 ug/ml leupeptin
1mM PMSF

3) Add 20ul Quick CIP (NEB #M0525; https://www.neb.com/products/m0525-quick-cip#Product%20Information) per 400ul of cell extract.

4) Incubate with Quick CIP at 37C for 1 hour.

5) Add 20ul lambda protein phosphatase (CST

Hypoxia Signaling

Learn here about the HIF-1 signaling pathway and its impact on oxygen homeostasis and cellular adaptation. Click here.

Exceptional Specificity of XP® Monoclonal Antibodies

Case Study 1: Exceptional Specificity of XP Monoclonal Antibodies pertaining to eXceptional Monoclonal Technology (XMT) from Cell Signaling Technology.

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Unfortunately, our PD-1 or PD-L1 antibodies have not been tested in neutralization assays. Therefore, we cannot guarantee their performance.


 

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We observe multiple bands at or around 33-35 kDa in cells with high STING protein expression, such as THP-1 and HDLM-2. Regrettably, we are not certain about what the lower molecular weight band represents in the THP-1 extract. However, customers may be interested in the following possibilities:

1) Please see Wang, P. et al. (2018) Nucleic Acids Res. 46, 4054-4071 (PMID: 29547894; https://pubmed.ncbi.nlm.nih.gov/29547894/). The two bands in THP-1 extract may represent the alpha and beta isoforms of STING.

2) Please see Rodríguez-García, E. et al. (2018) Immunohorizons 2, 363-376 (PMID: 31026807; https://pubmed.ncbi.nlm.nih.gov/31026807/). STING isoforms 2 and 3 do not include #13647’s epitope. However, STING isoform 1 may be detected by #13647.

3) Given that the lower band is also observed in 293T cells transfected with a construct expressing human STING protein (see image on the product webpage; https://www.cellsignal.com//products/primary-antibodies/sting-d2p…

Hypoxia and Cancer – the role of HIF-1α in oxygen sensing, metabolism, and tumorigenesis

Molecular oxygen is an essential element for metazoan life. Among its many roles, it functions as the final electron acceptor (oxidizing agent) during oxidative phosphorylation, a metabolic chain-reaction that generates energy in the form of ATP. HIF-1 alpha

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The methods for the LC and MS are shown below:
 

 ParameterSetting
Sample prepIP sample resuspension volume20 uL
 Sample volume per injection4 uL
 

Better IHC Intro and Step 1: Antigen Retrieval

Adhesion/ECM/Cytoskeleton

Overview of Adhesion and Extracellular Matrix signaling networks, antibodies and related reagents, interactive pathway diagrams, and technical resources.

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Our protocol is as follows:

REAGENTS:
TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174
Lipopolysaccharides (LPS) #14011

DAY 1:

  1. Seed THP-1 cells at approximately 8 X 10^5 cells/mL in 15 cm plates with 20 ml RPMI +10% FBS + 0.05 mM 2-mercaptoethanol. (We use plates instead of flasks since the cells adhere to the plate after treatment with TPA).
  2. Treat plates overnight with 80nM TPA.

DAY 2:
  1. Wash treated plates with 10 mL 1x PBS, drain, and add fresh media (RPMI +10% FBS + 0.05 mM 2-mercaptoethanol).
  2. Allow cells to rest overnight.

DAY 3:
  1. Remove media from plates.
  2. Add 15 mL serum-free RPMI to plates.
  3. Treat plates with LPS at 1ug/mL for 8 hours.
  4. Collect media after 8 hours at centrifuge at 1500 - 2000 RCF for 5 minutes.
  5. While the media is spinning, wash the cells from the TPA/LPS treated plates with 1X PBS and lyse in 1X SDS/D…

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The calculated molar extinction coefficient of rabbit IgG at 280 nm is approximately 210,000 M-1cm-1.

Chasing Tau-biomarkers for Alzheimer’s Disease [Part 1]

There are no tools or therapies to cure, prevent, or slow the progression of AD. Current therapeutic approaches include targeting amyloid plaques and neurofibrillary tangles.

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We recommend that you complete the BrdU cell proliferation assay as soon as you have the time. However, if you are unable to complete the experiment during the recommended time, you can complete the experiment through Step B: BrdU Incorporation and then pause. To do this, please follow the steps below:
1. Add the fixing/denaturing solution and incubate for 30 minutes (Part C: BrdU Assay Step 1).
2. Remove your solution and dry your plate at room temperature for 30 minutes. 
3. Cover plates with parafilm and store at 4C.
It is important to make sure your plate is completely dry before you cover with the parafilm and store it. Dry plates can be stored for up to 1 month at 4C, however, the signal of the assay may decrease slightly.

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Our Cleaved PARP (Asp214) (D64E10) XP Rabbit mAb #5625 is designed to be specific for the large fragment (89 kDa) of human PARP1 protein that is produced by caspase cleavage at Asp214. As such, it specifically recognizes an epitope at the N-terminus of the large fragment that only becomes exposed upon caspase cleavage. Therefore, #5625 will not recognize the c-terminal PARP-1 fragments (55 and 42 kDa) that are generated during necrosis by lysosomal proteases [see Gobeil, S. et al. (2001) Cell Death Differ. 8, 588-94 (PMID: 11536009; https://www.ncbi.nlm.nih.gov/pubmed/11536009)].
Unfortunately, we do not offer a PARP-1 antibody that will recognize both of the c-terminal PARP-1 fragments (55 and 42 kDa). Our PARP Antibody #9542 will not recognize either fragment; whereas, our PARP (46D11) Rabbit mAb #9532 may recognize the 55 kDa fragment, but not the 42 kDa fragment. 

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Both our AIF Antibody #4642 and our AIF (D39D2) XP Rabbit mAb #5318 are predicted to detect full-length AIF [amino acids 1-613 (predicted to be 67 kDa)], mitochondrial AIF [minus amino acids 1-54 (predicted to be 62kDa) and anchored to the inner mitochondrial membrane], and soluble/mature AIF [minus amino acids 1-101 (predicted to be 57kDa) and released to the cytoplasm to be translocated to the nucleus]. Although the apparent molecular weight is reported as 67kDa, the dominant band shown in our western blot images most likely represents mitochondrial AIF (predicted to be 62 kDa). Regrettably, the western blot images shown on the product webpages/datasheets do not include extracts prepared from apoptotic cells. However, multiple publications show detection of mature AIF by these antibodies.

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The western blot stripping/re-probing protocol for our Western Blot Stripping Buffer (5X) #91925 is below. However, it is important to note that we typically advise against stripping/re-probing because stripping can be incomplete and/or lead to inconsistent results through the loss of target proteins from the membrane. If possible, it is much better to run fresh immunoblots with each individual antibody on duplicated sets of samples. 

A.    Solutions and Reagents 
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

Supplied Reagents:
1. Western Blot Stripping Buffer (5X) #91925: To prepare 20mL of 1X Stripping Buffer, combine 4mL 5X Stripping buffer with 140uL 2-mercaptoethanol (0.1M final concentration) and 15.86mL RODI water.

Additional Reagents (Not Supplied):
1. 10X Tris Buffered Saline with Tween® 20 (TBST) #9997: To prepare 1 L…

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Freezing Collagenase at -20C will have no adverse effect on the enzyme.  Please make sure to allow the bottle to equilibrate to room temperature before opening to avoid the introduction of moisture from the temperature change.
 

PTMScan® Antibody Kits

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The NDRG1 target has 3 isoforms produced by alternative splicing (see Q92597). Isoform 2 and isoform 3 are missing amino acids 1-66 and amino acids 1-81, respectively. Depending on the species and cell line/tissue, each of the isoforms could be expressed at varying levels. Different treatments can also lead to varying amounts of isoform expression.

The respective antigens for NDRG1 (D8G9) XP® Rabbit mAb #9485, NDRG1 (D10F8) Rabbit mAb #9395, and NDRG1 Antibody #5196 correspond to residues in the carboxy terminus of human NDRG1 protein. Therefore, #9485, #9395, and #5196 can detect all 3 isoforms, leading to the observation of multiple bands. The antigen for NDRG1 (D6C2) Rabbit mAb #9408, on the other hand, corresponds to residues near the amino terminus and this antibody can only detect isoform 1. 
 

Four Steps to Better Immunohistochemistry: Step 1 - Antigen Retrieval

Companion reagents influence the binding of a primary antibody to its epitope dramatically affecting results. Learn IHC troubleshooting tips in this series

PTMScan® Motif Antibody Kits

PTMScan Motif Antibody Kits from Cell Signaling Technology

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The specificity of our 4E-BP1 (53H11) Rabbit mAb #9644 has been confirmed as follows:

1) See “Western blot analysis of extracts from control HeLa cells (Lane 1) or HeLa cells with a targeted mutation in the gene encoding 4E-BP1 (Lane 2)” on the product webpage at https://www.cellsignal.com/products/primary-antibodies/4e-bp1-53h11-rabbit-mab/9644.

2) 4E-BP1/2 DKO MEFs 

PMID: 28918902 (https://pubmed.ncbi.nlm.nih.gov/28918902/) Figure 6E