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Our ELISA kits are compatible with our RIPA Buffer (10X) #9806, however, this buffer may not work for some targets. RIPA buffer is very stringent in nature and can prevent the antibody/antigen from binding. For optimal performance, we suggest using the buffer included in the kit to generate your extracts. If you are generating tissue lysates, RIPA Buffer will better solubilize the proteins in the tissue compared to our Cell Lysis Buffer (10X) #9803 or PathScan^® Sandwich ELISA Lysis Buffer (1X) #7018.

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Unfortunately, we have not internally validated the use of acidic samples with our Cyclic AMP XP(R) Assay Kit #4339. We have had customers try this method, preparing their samples with 0.1M HCL and then neutralizing. The customers did not have success with this method.

If using acidic sample preparation, we recommend further diluting samples in 1X PBS after neutralization, prior to adding samples to the plate. Again, as we have not internally validated this, we cannot guarantee this kit will work with acidic samples.

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Please refer to following references for α-Synuclein:
PMID: 27322773 (https://www.ncbi.nlm.nih.gov/pubmed/27322773) Supplemental Figure S1 C - shows detection by western blot with clone D37A6 of high-molecular α-synuclein species after nonreducing SDS-PAGE.
PMID: 31370680 (https://www.ncbi.nlm.nih.gov/pubmed/31370680) Figure 4B - shows the specificity of clone D37A6 for mouse α-Synuclein. Clone D37A6 does not detect recombinant human α-Synuclein. Moreover, it does not detect a signal in samples prepared from α-synuclein knockout (KO) mice.
The antigenic peptide used to produce clone D37A6 shares 84% sequence identity with the human α-Synuclein ortholog.

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KinomeView Profiling is a western blot based screening technique used in conjunction with motif antibodies to optimize conditions (time, dose, cell line, compound) for maximum differential response. This can then be used to configure a follow-up PTMScan study with the appropriate motif antibodies.

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Our Fc Block CD16/CD32 (2.4G2) Rat mAb #88280 binds to the Fc receptor on the surface of live cells and blocks the interaction of the Fc receptor with the Fc domain of antibodies. Preventing this interaction ensures that your antibodies will bind only to their intended target, rather than being sequestered by Fc receptors.

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Yes, our DYKDDDDK Tag antibodies will detect 3X FLAG tag as long as the FLAG tag sequence is located on the N-terminal or C-terminal end.

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You can QC your CUT&RUN assay prior to NG-seq by performing qPCR analysis of your enriched DNA. Our CUT&RUN Assay Kit #86652 is compatible with downstream qPCR analysis.

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It is not uncommon to see a doublet with our beta-Actin antibodies depending on the samples being tested. It is likely caused either by phosphorylation of beta-Actin on its many phosphorylation sites, resulting in a shift in size, or by cross-reactivity with gamma-Actin (beta-Actin = 375 amino acids, gamma-Actin (cytoplasmic form) = 333 amino acids).

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Even though EGR1 has a predicted size of ~58kDa, 75kDa is the correct running size. This is because EGR1 is Sumoylated, which along with other PTMs, increases the size it runs at on a western blot.

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Based on homology, our β-Tubulin Antibody #2146, β-Tubulin (9F3) Rabbit mAb #2128, β-Tubulin (D3U1W) Mouse mAb #86298, and β-Tubulin (D2N5G) Rabbit mAb #15115 should detect β-Tubulin isoforms A, B, and C. The antigen sequences for these antibodies have 96% homology with beta 2C, beta 4B, beta 5B, and beta 4A isoforms 1, 2, and 3, and 95% homology with beta 2C isoforms A and B. Therefore they are likely to detect these subtypes.

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After the final purification step, we typically observe NGS libraries with a concentration ranging between 10-30 ng/ul. If the library DNA yield is too high, less PCR cycles can be considered.

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Cell lines replicate at different rates, therefore it is difficult to estimate a cell number to seed for optimal density for particular cell lines. On the XTT Cell Viability Kit #9095 datasheet, we show a range of densities from ~1500 cells/well - 100K cells/well and their associated signal intensities. At 100,000 cells per well, we start to see a plateau. We recommend performing a density titration similar to the one we show on our data sheet in order to determine the optimal concentration for your final assay. In terms of incubation time, this depends on your experimental conditions. If testing compounds, incubation times can vary from hours to days. It is necessary to adjust your seeding density to account for the additional days the cells will be in culture.

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No materials of animal origin are used in the production of our Human IL-22 Recombinant Protein #74808. Therefore, additional testing for infectious material is not relevant.

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The antigen for our Rab5A Antibody #2143 corresponds to residues surrounding Gly190 of human Ras-related protein Rab-5A (UniProt ID P20339). The antibody is predicted to cross-react with endogenous levels of Ras-related protein Rab-5A, but is not expected to cross-react with Ras-related proteins Rab-5B or Rab-5C.

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The GFP Antibody #2555, GFP (4B10) Mouse mAb #2955, GFP (5G4) Mouse mAb #55494, and GFP (D5.1) XP® Rabbit mAb #2956 are not expected to detect Turbo GFP due to low sequence homology.

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The IFN-α (6B18) Mouse mAb #3110 and IFN-α (8C21) Mouse mAb #3115 antibodies are different clones to the same target. These antibodies are approved for use with recombinant protein and will not detect endogenous levels of IFN-α. While either antibody will work well, #3110 is slightly stronger than #3115 by western blot and in addition has some citations in CiteAb.

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The CUT&RUN Antibody Binding Buffer #15338, CUT&RUN 10X Wash Buffer #31415, and diluted CUT&RUN 1X wash buffer are stable at RT or 4C for one month. However, the Digitonin Solution #16359, Spermidine #27287, and Protease Inhibitor Cocktail #7012 can start to lose activity quickly if not stored at -20C. For the best performance, we recommend adding these components to the CUT&RUN Antibody Binding Buffer or CUT&RUN Wash Buffer on the day of the experiment and keeping them on ice during use.

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The antigen for the β1-Adrenergic Receptor Antibody #12271 surrounds Gly406 of the human β1-Adrenergic Receptor protein and is contained entirely in the cytoplasmic domain. #12271 detects full-length β1-Adrenergic Receptor as shown on the datasheet (see western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human β1-Adrenergic Receptor (β1AR; +). The smeary bands that can be observed above 50 kD are a result of N-glycosylation. We have not looked into the issue for further details.

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The SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit #69486 has not been validated for use with any samples except for human serum or plasma. However, it may also be used to assess the blocking activity of non-human antibodies and small molecules. For example, we have had customers successfully use this kit in rat samples. You will likely need to optimize the sample dilution and antibody spike-in concentrations, and we recommend running appropriate controls for each assay (for example, rat sera that should not block activity or concentration-matched, non-relevant antibodies that are spiked in).

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Each GTPase Assay Kit provides enough reagents for 30 immunoprecipitations based on the amount of GST-fusion protein, Spin Cups, and Collection Tubes provided. The GTP and GDP is only enough for 10 assays. These two reagents are intended to be used as positive and negative controls alongside your own samples. For example, if working with a cell line treated with a compound to see if it would induce active Ras signaling (when using our Active Ras Detection Kit #8821, specifically), one would want to run these samples with GTP + GST-Raf1-RBD (positive control), GDP + GST-Raf1-RBD (negative control), and the samples + GST-Raf1-RBD to see if the treatment effectively induced active Ras compared to the positive control. In other words, GTP and GDP are usually not added to all 30 assays; 10 assays worth is typically an appropriate amount of these controls.

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The Senescence β-Galactosidase Staining Kit #9860 is intended to be a qualitative assay to determine if cells are senescent. We have not determined a means of quantification in-house. However, we usually direct customers to the following paper where Hoechst (#4082) was used as a DNA dye to quantify cells in frozen tissue:

Baker, D. J. et al. (2004) BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nat. Genetics 36, 744-9 (PMID: 15208629; https://pubmed.ncbi.nlm.nih.gov/15208629/)

The paper below is another we frequently send to customers interested in quantifying their samples:

Gary, R.K. and Kindell, S.M. (2005) Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts. Anal Biochem 343(2), 329-34 (PMID: 16004951; https://pubmed.ncbi.nlm.nih.gov/16004951/)

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HPSE is synthesized as a 65 kDa inactive precursor that undergoes proteolytic processing, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. The 65kDa proform and the 50 kDa subunit (amino acids 158-543) are detected by the HPSE (E3N3H) Rabbit mAb #99756, while the 8kDa subunit (amino acids 36-109) is not.

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We have tested the Griess Reagent Nitrite Measurement Kit #13547 with media, cell culture supernatant, and urine. It should cross-react with all species types.

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Our Akt Antibody #9272 detects all 3 isoforms of Akt: Ak1, Akt2, and Akt3.

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The Cellular Glutathione Detection Assay Kit #13859 detects only the reduced form of glutathione. It is unable to detect the oxidized form.

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We do not recommend using multiple unconjugated antibodies from the same species in a flow cytometry experiment because it will not be possible to detect each antibody target independently. If your experiment requires the use of multiple antibodies from the same host species, we suggest using antibodies that are directly conjugated to a fluorophore. If a fluorescent conjugate for your antibody of interest is not available, consider using our custom conjugation services to create a solution that will fit the needs of your experiment.

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Both enzymatic digestion and sonication work when performing ChIP for histones, transcription factors, and cofactors. However, we have found that enzymatic ChIP gives higher levels of enrichment than sonication ChIP for transcription factors and cofactors, especially those with low expression. We have also found that some transcription factors and cofactors only work in enzymatic ChIP and not sonication ChIP. That being said, sonication ChIP kit has its advantage when dealing with fibrous or fatty tissue types. Please refer to our online video (https://www.cellsignal.com/learn-and-support/videos-and-webinars/webinar-enzymatic-or-sonication-protocol) for which kit to choose for your ChIP experiment.

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All of our siRNAs are single duplexes. We discontinued pools a number of years ago to avoid off-target effects.

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Yes, the Fc-Tag should be able to be purified with Protein A or G beads.