Identify and quantify membrane proteins in a total proteome study for a more complete view of proteins on the cell surface.
The antigen for our Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) Antibody #4228 shares 100% sequence homology with human. However, we have tested many different human samples in-house and have not been able to obtain signal with this antibody.
The molecular weight reported by Simple Wesetern™ instruments like Jess™ is an "apparent" molecular weight as opposed to an "exact" molecular weight. The molecular weight of the target is estimated based on calibration to the protein ladder and internal standards.
Discover the range of Alexa Fluor 488 Conjugated Antibodies at CST for superior fluorescence in your research. Explore options for a variety of targets. Click here.
For western blotting, we recommend mixing the primary antibody in the appropriate dilution buffer (see product datasheet) with at least 1.0 ug of peptide per 1.0 ul of primary antibody and incubating for 20 minutes with agitation before adding the mixture to the membrane. For example, in 10 ml of primary antibody dilution buffer with 10 ul antibody (1:1000 final dilution), you would add 10 ul peptide stock that is at 1.0 mg/ml (1:1000 final dilution).
The Calpain 2 Large Subunit (M-type) Antibody #2539 detects both active and inactive Calpain 2, which migrate at the same size (79-80 kDa). When Calpain 2 is autoproteolytically cleaved at Ser20, it can result in a 78 kDa band.
We have found that a doublet may appear if samples are boiled prior to gel loading when performing a western blot with our Na,K-ATPase Antibody #3010. The protein structure of this target is extremely sensitive to denaturing elements such as temperature, pH, ionic strength, presence of native lipids, cholesterol levels, and detergent concentration.
Streamline Targeted Protein Degradation Drug Discovery with specific, application-validated antibodies and industry-leading proteomic services.
We have several antibodies for detecting β-actin that are validated for IF-IC with human samples. These include: β-Actin (13E5) Rabbit mAb #4970β-Actin (D6A8) Rabbit mAb #8457β-Actin (8H10D10) Mouse mAb #3700β-Actin (E4D9Z) Mouse mAb #58169. All of these antibodies work well and there is no preference for this application.
A CUT&RUN assay allows signal normalization among samples or among experiments by adding spike-in DNA in the stop buffer. Since you add the same amount of spike-in DNA in the assay, the signal from spike-in DNA should be the same among all samples or experiments.
The Bcl-xL (54H6) Rabbit mAb #2764 recognizes a portion of the human Bcl-xL sequence which is retained in all three isoforms of Bcl-xL. Please see UniProt for the precise isoform sequences:https://www.uniprot.org/uniprot/Q07817#sequencesThe doublet is most likely Bcl-xL and Bcl-xB, as Bcl-xS has a lower predicted molecular weight (19 kDa versus 25-26 kDa).
Our Normal Rabbit IgG #2729 is an unconjugated rabbit polyclonal antibody that is routinely used as a non-specific rabbit IgG control for IP and ChIP, whereas our Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 is an unconjugated rabbit monoclonal antibody that is routinely used as a non-specific rabbit IgG control for IP, IHC-P, IF-IC, F, and ChIP.Which of the two is recommended depends on the application:IP:#3900 is recommended as a negative control for IP experiments with rabbit monoclonal antibodies, whereas #2729 is recommended as a negative control for IP experiments with rabbit polyclonal antibodies.IF and Flow: #3900 is recommended as a negative control for IF and flow cytometry experiments with both rabbit polyclonal and rabbit monoclonal antibodies.IHC:#3900 is recommended as a negative control for IHC experiments with rabbit monoclonal antibodies, whereas Jackson ChromPure IgG (#011-000-003; https://www.jacksonimmuno.com/catalog/products/011-000-003) is recommended as a negative control for IH…
This video demonstrates the technique used for splitting adherent cell lines with trypsinization.
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).
CST thoroughly validates each antibody available in the SignalStar Multiplex IHC Panel Builder menu. Various combinations of antibodies are tested through titration and fluorophore pairing, and in both rounds of imaging.
SignalStar Multiplex IHC Kits and reagents are optimized and validated, but if you do have an issue, this troubleshooting guide can help.
Watch the webinar series Antibody Application Insights and enhance your results in antibody-based applications such as Western Blot or Immunofluorescence. Gain valuable insights, tips, and tricks through our webinar series, and take your research results
It is not uncommon for Akt to appear as a doublet on a western blot. This double banding is due to differential phosphorylation of the different Akt isoforms.
Based on sequence homology, we predict that our α-Tubulin Antibody #2144 and α-Tubulin (11H10) Rabbit mAb #2125 will detect the alpha 1a, alpha 1b, alpha 1c, alpha 2, alpha 3c, alpha 3d, alpha 3e, alpha 4a, and alpha 4b subtypes.
An overview of oh mass spectrometry-based proteomics studies can be a powerful tool for drug discovery and development
For imaging purposes, we recommend using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb #9664 when working with rodent tissue. We have noted non-specific labeling in specific sub-types of healthy cells in fixed-frozen mouse tissues (e.g. pancreatic alpha-cells) and nuclear background in rat samples with Cleaved Caspase-3 (Asp175) Antibody #9661 and Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb #9579.
Our tissue extracts are prepared as follows: Cut a small piece of tissue (approximately 3 mm^3).Place in a clean Dounce homogenizer.Add 1mL cold 1X Cell Lysis Buffer #9803 + 1.0 mM PMSF #8553 + 1X Protease/Phosphatase Inhibitor Cocktail #5872.Homogenize well (approximately 20 strokes).Sonicate 3 times for 15 seconds each (cool on ice in between).Microcentrifuge at 12,000 rpm for 15-20 minutes to remove insoluble debris.Add 333uL of 3X SDS Sampler Buffer (adjust as needed depending on how much 1X Cell Lysis Buffer you added) or store at -80C in 1X Cell Lysis Buffer. Notes: Keep unused tissue on dry ice.Perform all steps on ice.3X SDS Sample Buffer can be prepared using the Blue Loading Pack #7722 or Red Loading Pack #7723. To prepare fresh 3X Sample Buffer, add 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.Tissue lysates have short shelf-lives and should be made fresh frequently.
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 FastScan kits differ from the PathScan kits in that (1) they come with a positive control and (2) the assays can be performed much faster. With FastScan, the samples are incubated with a capture antibody conjugated with a proprietary tag and a detection antibody linked to HRP with no need for wash steps.
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.
Regrettably, we cannot offer any firm guarantees when glycerol formulated antibodies have been frozen. However, we have performed stability testing by western blot for a representative group of rabbit polyclonal, rabbit monoclonal, and mouse monoclonal antibodies, and found that two freeze/thaw cycles on dry ice (-78.5C) did not affect their activity.
