What are the Ficoll-Paque Plus / Premium protocols for isolating lymphocytes from animal blood (cow, calf, horse)?
The Ficoll-Paque products are developed for the preparation of mononuclear cells from human sources with a density of 1.077 g/mL. Ficoll-Paque Premium is also available in densities 1.073 g/mL and 1.084 g/mL for the preparation of different density mononuclear cells: lower density human mononuclear cells, for example mesenchymal stromal cells (MSCs) or monocytes (1.073) or higher-density human mononuclear cells from blood and bone marrow (1.084). The latter can further be used for separating blood cells from mice or rats.
Other types of animal cells do not have any in-house protocols for using Ficoll-Paque Plus/Premium, but you can find some references in our handbook. Isolation methods using Ficoll-Paque for preparation of mononuclear cells have been described for mouse, dog, monkey, cow, rabbit, horse, pig, and even fish.
There is also another option, Percoll, our density gradient media for cell centrifugation, that you might find interesting. It is not a ready-to-use solution, so you need to create the gradient yourself. See more in our Isolation of Mononuclear Cells handbook about how to best use it.
Can I use NAP columns to purify proteins? What is the maximum loading capacity for NAP columns in milligrams (mg)?
You can use NAP columns for protein purification, oligo purification, desalting, and buffer exchange. NAP columns contain Sephadex™ G-25 Medium DNA Grade. DNA Grade Sephadex is tested to ensure reproducibility and high recovery of DNA (>90%) specifically. This does not mean that they would not be suitable for proteins as well. The corresponding products for gravity flow protein purification, the PD columns, contain Sephadex G-25 Medium. These are not tested for DNA, only for protein.
NAP and PD columns work in the same way using gel filtration (size exclusion chromatography), where molecules are separated based on differences in size. Molecules larger than the largest pores in the Sephadex matrix are excluded from the matrix and are eluted first. It is the sample volume that is critical, not so much the concentration.
Note: Within the PD range we also have columns with Sephadex™ G-10 with a lower MW (molecular weight) cut off.
The signal on my ECL detection kit is short lived/fading. What do you recommend?
Achieving the correct concentration for the antibodies is very important; if the antibodies are too concentrated it may cause signal fading. Too much HRP (HorseRadish Peroxidase) (HRP) can “eat-up” the substrate too quickly and at the end the signal captured is low.
If you don’t see any signal at all, it may also be due to a problem with the substrate itself. A quick way to check it is to perform the blue light test as follows:
- In a dark room, prepare 1 to 2 mLl of detection reagent working solution in a clear test tube.
- Add 1 μLl of undiluted HRP-conjugated antibody solution.
The solution should immediately emit a visible blue light. If not, it suggests that the substrate is not working (this may be due to cross-contamination between the contents of the 2two bottles or a degraded substrate).
How can I decrease background in my Western blotting?
If you see a spotty, uneven background, it may be due to some areas of the blot drying during some of the incubations.In general, high backgrounds are linked to:- Non-optimal choice of antibody dilutions (both for primary and secondary antibodies)
- Low amount of target protein, leading to long exposure time
- Contaminated buffer or not perfectly clean blotting equipment
- Insufficient blocking; for example, the concentration of blocking agent may be too low, it may not be the best type of agent for the application, or it may not be a freshly prepared solution
- Insufficient washing, including number of washes, time, volumes used, Tween concentration, insufficient movement of the washing buffer over membrane
- Overexposure of film
Note: We can help you troubleshoot your Western blotting assay. Please contact our Support Scientists by filling out the form at the bottom of the page.
How can I increase sensitivity in my Western blotting in six steps?
There are several aspects you can optimize to increase the sensitivity of Western blots:
- Choice of membrane:
- If you are using NC (NitroCellulose (NC)), you may want to switch to polyvinylidene difluoride (PVDF), which is more resistant if re-probing is necessary.
- If your proteins are small (< 20 000 Da (Dalton) or even < 10 000 Da you may need to use smaller membrane porosity: 0.2 µm)
- You may also need to optimize the transfer time.
- You can reduce the antibody dilutions and increase the probing time, especially for the primary antibody.
- Switch to a higher sensitivity substrate: if you use chemiluminescence, you may not be aware of the whole range of substrates we offer with increasing sensitivity: ECL start, ECL, ECL Prime, ECL Select.
- Be sure you use the right films. Our Hyperfilm ECL is optimized for chemiluminescence and are completely transparent, offering an ideal signal-to-noise ratio.
- You can also increase the exposure time and try different exposures to find out the best one.
Chapter 10 of our Handbook is completely dedicated to Western blotting troubleshooting.
Contact our Support Scientists by filling out the form at the bottom of the page, should you have any further questions.