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We typically perform this assay using the Senescence β-Galactosidase Staining Kit (#9860) from start to finish in one day and have not paused between fixing and staining. Therefore, we cannot guarantee the results will not be negatively impacted if cells are incubated in PBS buffer after fixing instead of immediately proceeding to overnight staining. However, we have spoken with numerous customers who have paused after fixing, stored their cells in PBS at 4C for a few days prior to staining and have had great success.

Resources

We have several antibodies for detecting the phosphorylation of ribosomal protein S6 (rpS6) at Ser235/236 that are validated for IHC-P with human samples. These are S6 Ribosomal Protein (5G10) Rabbit mAb #2217 and S6 Ribosomal Protein (54D2) Mouse mAb #2317. These antibodies perform similarly for IHC-P with human samples, therefore, there is no preference for this application.

Writing a Grant Part 3: The Experimental Approach

This section expands on the solution and illustrates your research plan—but don't forget to address potential pitfalls and offer alternative approaches!

Resources

Histones have numerous post-translational modifications (PTMs), which can cause the protein to run at a different rate during electrophoresis. The upper band that migrates at ~25-27 kDa is likely phosphorylated histone H2A.X which also contains additional PTMs, such as ubiquitination. We believe this to be the case as the signal of the phosphorylated histone H2A.X band at 15 kDa and the upper band at 25-27kDa both increase in signal strength upon UV treatment. In addition, the 25-27kDa upper band can be seen with our total histone H2A antibodies (#7631, #2595, #50722), in both untreated and treated samples, indicating that it is specific to histone H2A.

CAMK Protein Kinases

A kinome diagram showing the CAMK group of protein kinases from Cell Signaling Technology.

Atypical Protein Kinases

Kinome diagrams showing the atypical protein kinase groups from Cell Signaling Technology.

Something to YAP About! Progress in Targeting YAP/TAZ-TEAD Signaling in Cancer Research

Breakthroughs in therapeutic targeting of YAP/TAZ-TEAD signaling using small molecules were presented at AACR 2023. Read a recap of the presentations.

CST Takes Leadership on UN COP21 Climate Targets

Cell Signaling Technology (CST) is proud to announce a plan to reduce our greenhouse gas (GHG) emissions to meet voluntary targets set by the 2015 COP21 Paris Climate Accords.

Resources

We have not attempted RNA isolation using the Cell Fractionation Kit #9038, as this is not the intended application. The buffers and protocol (centrifugation times, speed, temp, etc.) have not been optimized for RNA and none of the buffers are RNase-free. In addition, for RNA isolation, cell pellets need to be subsequently treated with TriZol, chloroform, and isopropanol. Even if the fractionation buffers were RNase-free, it is difficult to speculate if any components could possibly interfere with RNA isolation. RNA isolation is sensitive and can be tricky, therefore I would not recommend complicating the RNA extraction/isolation process by simultaneously fractionating cells for protein. There are separate RNA Subcellular Isolation Kits commercially available.

LIM Protein Domain

A scientific resource for the LIM protein domain containing information on structure, function, and domain binding to mediate protein-protein interactions.

Unraveling the Role of G3BPs in the Regulation of Cellular Energetics

Scientists used around 30 different Cell Signaling Technology® antibodies to characterize the Ras GTPase-activating protein-binding proteins G3BP1 and G3BP2.

Resources

We recommend using our CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 to make DNA libraries for CUT&Tag. Our DNA Library Prep Kit for Illumina (ChIP-seq, CUT&Tag) #56795, and its paired Multiplex Oligos for Illumina (Dual Index Primers) (ChIP-seq, CUT&Tag) #47538 or Multiplex Oligos for Illumina (Single Index Primers) (ChIP-seq, CUT&Tag) #29580 are not compatible with CUT&Tag DNA samples.

MH2 Protein Domain

A scientific resource for the MH2 protein domain containing information on structure, function, and domain binding to phospho-serine motifs during TGF-beta signaling.

FERM Protein Domain

A scientific resource for the FERM protein domain containing information on structure, function, and domain binding in cytoskeleton-associated proteins.

Writing a Grant Part 7: How to Interpret Summary Statement and Reviewer’s Critiques

After all your hard grant writing work, now it's time to see if you've secured funding or if it's time to go back to the drawing board.

GYF Protein Domain

A scientific resource for the GYF protein domain containing information on structure, function, and domain binding to proline-rich motifs.

GRAM Protein Domain

A scientific resource for the GRAM protein domain containing information on structure, function, and phospholipid binding during vesicle trafficking.

Resources

At this time, we have no data that suggests the expression platform matters for protein performance. Typically, the difference between expression systems is yield rather than performance. The main difference between these products is that the domains of the two proteins are different. The Human ACE2 (18-615) Recombinant Protein (hFc-Tag) #38365 spans amino acids 18-615, whereas the Human ACE2 (18-652) Recombinant Protein (hFc-Tag) #99339 spans amino acids 18-652. There is evidence suggesting that 18-652 is at least partially cleaved where 18-615 is not (this appears in their respective datasheets in the purity images). For that reason, depending on your assay, the Human ACE2 (18-615) Recombinant Protein (hFc-Tag) #38365 may be more appropriate. 

Still in the Dark: AMD Patients Wait for Answers

Our summary of recent findings published in Nature, which indicate that non-performing antibodies were used in many recent macular degeneration studies.

Resources

The amount of CUT&RUN DNA required for NG-seq analysis depends on the sensitivity of the DNA library prep kit. The typical yield for CUT&RUN DNA is 0.6 to 6 ng per reaction. Our SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795 will work with starting DNA as low as 0.5 ng. You can also increase the number of PCR cycles up to 20 to increase amplification of the library if the starting amount is too low.

Resources

Due to the fact that the total α-Adducin (D7T7R) Rabbit mAb #70174 does not recognize this band in our in-house testing, the 150kDa background band seen with our Phospho-α-Adducin (Ser12) (E5X8Y) Rabbit mAb #84214 is likely an unrelated protein induced by nocodazole.

FF Protein Domain

A scientific resource for the FF protein domain containing information on structure, function, and binding to phospho-serine motifs.

Resources

The Cre Recombinase (D7L7L) XP Rabbit mAb #15036 has a "recombinant" sensitivity. Therefore, it can detect Cre in transgenic mouse models. In contrast, the Cre Recombinase (D3U7F) Rabbit mAb #12830 has a "transfected only" sensitivity because it has been validated using only transfected Cre recombinase; it does not detect Cre in transgenic mouse models.

Resources

Concanavalin A beads can clump together rather easily, especially when cells become lysed. Make sure your cells are healthy and be sure to treat them very gently during the cell wash. We also recommend limiting the room temperature incubation time of the cell suspension with Concanavalin A beads to 5 minutes. Resting the tubes at the appropriate incubation temperatures in lieu of rotating or rocking the tubes during the incubations for cell-bead binding, antibody binding, and pAG-Tn5 enzyme binding helps to mitigate bead clumping and does not have any adverse effects on sequencing depth, identified peak numbers, or binding patterns.

With all that being said, our internal testing has shown that bead clumping does not negatively affect the final results of CUT&Tag experiments.

EF-Hand Protein Domain

A scientific resource for the EF Hand protein domain containing information on structure, function, and calcium binding.

Resources

Even without DTT, non-reducing conditions are harsh on protein structure. In-house, samples are heated to 95°C for 5 min in the presence of SDS, so the protein (including the trimerization domain) is fully denatured. This results in a loss of its ability to trimerize. It is possible a native gel would run closer to the trimeric molecular weight. We have also run SEC-HPLC on this product to confirm that we observe the expected MW of a trimeric protein when it is in solution. 

C1 Protein Domain

A scientific resource for the C1 protein domain containing information on structure, function, and binding to phospholipids.

Using SignalScan to Investigate Infection and Immune Response

SignalScan bridges the gap between PCR-based detection of COVID-19 infection and uncovering the molecular signature of infection and host immune response.

Neurodegeneration: Tau Protein and Neurofibrillary Tangles

While the Tau protein is phosphorylated in normal, healthy brain, it becomes hyperphosphorylated in neurodegenerative diseases, like Alzheimer's.

Rooftop Solar Comes to CST

We're using solar power now at CST and you should too! We've taken steps to formally register our climate change goals with the “We are still in” campaign.