Methods

Dear June, I agree with Philipp, your insert may be too large for 6kb vector... Also, there is another possible problem - as I understand, you've used purified from agarose gel cutted vector. Procedure of cutting under UV should be performed with highest accuracy, because UV damage of DNA can lead to its damage. You can try to find topics about this on RG. Good luck!

Thanks! I actually found a publication by JB Kirkland (2011) that states that "the NAD/NADH redox couple participate in around 400 Rns, while that of NADP/NADPH participates in around 30 more" ("Present knowledge in nutrition" 10 ed. 2012).

Spin the tube and estimate the volume using a P1000 pipette. You really won't need much (5-10 ng) to transform the DH5. The plasmid is already prepared and you only really need 1 colony. It then would make sense to sequence the plasmid before making the virus. In this case its to be sure no replication error occurs, when getting plasmids from collaborators it makes sense in case they have sent the wrong plasmid (it happens). Before spending a lot of time and effort its best to be 100% sure you have what you think you have.

My bad, I misread the "maize" part of your message, yeas I agree as well with Daniel, ECR is just for vertebrates.

No experience ....But in accordance with the available literature, Mucin can be dissolved in100 mM Acetate Buffer (pH 5.0). Its solubility is 4 mg/mL and yields Hazy, Pale Yellow-Brown soln.

Review on characterizing ABeta by several methods including SDS-PAGE, Western Blot and ELISA, DLS, FCS,SEC. ESI-MS/MALDI and capillary electrophoresis

U Welcome...

Can u be more explicit? What sort of data u r looking for?

Try with LGC Genomics (ex KBiosciences). They offer the KASP genotyping system http://www.lgcgenomics.com/

Thats important to decide which type of surgical suture will be best ? Lipid ratio generally play major role for Pancreatic Fistula. I want to eliminate this undesired effect after Pancreatoduodenectomy.

Dr Ying-Xin Fan has identified all of the variables that concern myself when using WB for protein quantification.

Exactly it is! Thank you!

And for people who love mendley and can't install it on their university computers there is a portable version available which the creators sent me to test. It is not too bad and can atleast be used to organise Pdf of the computer. I will be happy to zip it and send it to anyone who needs it..

Thank you Dr. Azra for your kind information I am in USA for next 2 weeks

Nonidet is a compound based on PEG. When you will evaporate the solvent from this solution you will likely run into trouble with the sample viscosity as well as with PEG signals in the mass spec (as it will not be removed then. If you do ultrafiltration, you will get at least a part of the nonidet removed (as it is smaller than 3kDa). In this case I would definitely recommend ultrafiltration BUT: make sure you saturated the membrane with protein first (e.g. spin an aliquot of a BSA digest or something similar through it). Otherwise you might observe peptide/protein adsorption onto the membrane which reduces your sensitivity by sample loss. Alternatively, you can apply the FASP digestion from Matthias Manns group (sorry, can't find the reference right now). That facilitates the digestion directly on the membrane and spins the peptides through after digestion.

Rajbardhan, I've never given the MACS technique a shot with PMNs, although others in my lab have done it. They've hade issues getting the PMNs to work, as measured by migration, following purification by MACS. For my experiments, where I look for cytokine production, I've used a Percoll gradient centrifugation methods. Using this technique I get about 85% purity, and the lab I got the protocol from claims up to 95% purity. To avoid the high calcium/magnesium environments I've simply used HBSS (no Ca/Mg) as the diluent for the Percoll and as the buffer that I keep the PMNs in until I'm ready to plate them out. Once they're ready I'll get them into DMEM. Generally PMNs will last for about 6-8 hours unstimulated, and once they've been stimulated with something like LPS (endotoxin) they're life can be extended to about 12 hours. I've never done a PMN isolation from peripheral blood, rather I get my PMNs from the bone marrow (from the femurs and tibias) which will be approximately 15-25% PMNs (MHCII- Gr1+ CD11b+) by flow cytometry prior to enrichment. Using the Percoll method of PMN isolation from bone marrow I've gotten upwards of 1x10^7 cells per mouse. And as a heads up PMNs tend to be very finicky cells, so take care while isolating them. Best of luck!!

Endothelial cells cultivated under artifical tissue culture change a little biz phenotype according to used media, supplements and coating. So its easily possible that they express alpha actin.

I think the major area of interest is the way in which chromatin, DNA methylation and non-coding RNAs cooperate to control gene expression and cell phenotype.

You might be better remove the sovelable MTT in order to avoid the false positive OD.

The prerequisite for an ELISA is a highly purified antigen. If you have this, you can run a direct ELISA (solid phase: antigen GULT4 // sample with antibody // anti-species-enzyme labeled // substrate reaction // stop // measurement). If you have no purified antigen, you can use an indirect ELISA version with one incubation step more: (solid phase: anti-GLUT4-antibody // GULT4 crude antigen mixture //sample with antibody // anti-species-enzyme labeled // substrate reaction // stop // measurement). This requires an specific anti-GULT4 antibody either mono- or polyclonal. This antibody on the solid phase will catch the antigen only in the next incubation step. All other steps are comparable. You have to take care only, that your anti-species-antibody (labeled) is not cross reactive with the solid phase antibody. Anyhow, you need just on control well more on your plate. If you have once established your ELISA, you will be cheaper and faster than any Western blot. You will have another advantage: you can run more sample per experiment and you can introduce a calibration curve by using an antiserum/antibody and defining a given dilution as standard with e.i. 100 AU/mL. See figure 4 in the attached paper (indirect assay).

If it's not too late already, maybe have a look at the Ludin Chamber. Up to six fit into a rack: http://www.lis.ch/thechamber.html

what about microRNA in plasma? I wanna use mirCurry. What do you think about it?

I recommend the paper: "Use of spectroscopic probes for detection of reactive oxygen species" by G. Bartosz , Clinica Chimica Acta 368 (2006) 53 – 76

Yes normaly this works... It could be, that there are some differencenes in the total amount you get out of one prep, but in my experience you can change colums between different kits and also between different manufactors.

Both are chaotropic agents which destabilize H-bonds and other weaker bonds in macromolecules like DNA and they are precipitated out with salt. Isopropanol is a better chaotropic agent than ethanol and so the vol required for precipitation is much less. One has to remove these reagents for further processing and it is much easier to remove ethanol than isopropanol.