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Immunocytochemistry
Immunocytochemistry: Methods, Techniques and Protocols
Introduction, cell preparation, fixation, antibody staining, observation, references and useful links.
Western blot
Western protocol
Detailed western protocol and buffers preparation.
All about western blot
Protocols, troubleshooting, theory, buffers and definitions.
Western blot troubleshooting
High background signal, strange/non-specific bands on the blot, low sensitivity, is a right band detected?, uneven results with lots of
spots.
Cell culture and cell preparation
Fundamental Techniques in Cell Culture
Basic Techniques, Aseptic Technique and Good Cell Culture Practice, Ressuscitation of Frozen Cell Lines, Subculture of Adherent Cell
Lines, Subculture of Semi-Adherent Cell Lines, Subculture of Suspension Cell Lines, Cell Quantification, Cryopreservation of Cell
Lines, Testing for Bacteria and Fungi, Detection of Mycoplasma by Culture, Testing for Mycoplasma by Indirect DNA Stain.
Basics about Tissue Culture Methods
Types of cells grown in culture, work area and equipment, preservation and storage, maintenance, safety considerations, tissue culture
procedures.
Virus infection
Lentiviral strategies for RNAi gene knockdown
Introduction, Choice of lentiviral vector, Design of the short RNA hairpin, Cloning the short hairpin sequence into the lentivirus
plasmid, Production of short hairpin RNA virus, Viral Packaging, Using the virus, Examining the knockdown, Creation of transgenic RNAi
mice by lentiviral transduction, Literature cited.
Immunoprecipitation
General Principles of Immunoprecipitation
The choice of a lysis buffer (RIPA buffer, NP40 buffer, PBS, TN).
Immunoprecipitation (Boiling in SD, Antisera, Bugs, Secondary Antibodies, Cell Lysis, Pre-clearing).
Cell lysates
General lysis buffers
The choice of lysis buffer depends on what kind of experiment you are doing.
RIPA buffer gives the lowest background, but can denature some kinases. It also has the potential to disrupt protein:protein
interactions.
NP40 buffer (EVF) is less denaturing, but gives a higher background. It is less likely to inhibit kinase activity and disrupt protein
complexes.
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Additives:
EDTA prevents phosphorylation in the lysate. We usually use 2 mmolar.
Sodium vanadate inhibits all tyrosine protein phosphatases. We use 200 micromolar and add it fresh for each experiment from a stock
made in water and stored in a plastic tube at room temperature.
Sodim fluoride is an inhibitor of Serine/Threonine protein phosphatases. We use it at 50 mmolar.
These should be added to both RIPA and NP40/EVF.
Some people like to add Trasylol or aprotinin, a protease inhibitor.
In our experience, there is no need to prepare the vanadate any special way. Storage in glass leads to the appearance of precipitates.
Phosphate-buffered RIPA is generally the best choice. Phosphate is a good buffer at pH 7.2 and also functions as an inhibitor of
phosphatases. Tris is a poor buffer at pH 7.2 and does not inhibit phosphatases. Tris is helpful if you need to add calcium or
manganese, both of which will precipitate phosphate. Otherwise, phosphate is preferable.
RIPA is:
1% NP-40 or Triton X-100
1% sodium deoxycholate
0.1% SDS
0.15 molar NaCl
0.01 molar sodium phosphate, pH 7.2
1% Trasylol, a 1:100 dilution of what comes from Mobay
If Tris is substituted for phosphate, use 50 mmolar Tris-HCl, pH 7.2 .
Add sodium fluoride and EDTA. The EDTA is very important. The fluoride is less so. Add the vanadate freshly each time you use it.
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NP40 buffer used to be our name for RIPA lacking DOC and SDS. It is now called EVF, which refers to the additives, not the
distinguishing detergent. NP40 buffer can be made with either NP40 or Triton X-100, and can be buffered with either phosphate or Tris,
depending on your needs.
NP40 buffer is:
1% NP-40 or Triton X-100
0.15 molar NaCl
0.01 molar sodium phosphate, pH 7.2
1% Trasylol, a 1:100 dilution of what comes from Mobay
If Tris is substituted for phosphate, use 50 mmolar Tris-HCl, pH 7.2 .
Add sodium fluoride and EDTA. The EDTA is very important. The fluoride is less so. Add the vanadate freshly each time you use it.
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PBS, in the context of immunoprecipitation, is:
0.15 molar NaCl
0.01 molar sodium phosphate, pH 7.2
The PBS used in tissue culture is a more complex physiological phosphate- buffered saline which contains calcium, magnesium, and
potassium
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TN is:
0.15 molar NaCl
0.05 molar Tris-HCl, pH 7.2
Cell culture and cell preparation
Single cell cloning by serial dilution
This technique is widely used for clonal isolation of hybridomas and other cell lines that are not attachment dependent. However, it is
also very useful for cloning attachment dependent cells when the cell plating efficiency is very low, unknown or unpredictable. This
method is fast and easy.
PC12 cells
Growth of PC12 cells, poly-L-lysine coating of plates for PC12 secretion and transfections, harvesting PC12 cells for reporter assay,
Harvesting PC12 cells for reporter assay, Harvesting PC12 cells for reporter assay, Transfection of PC12 cells (Lipofectamine),
Transfection of PC12 cells (Superfect), Transfection of PC12 cells (Lipofectin), Transfection of PC12 cells (GenePorter).
Virus infection
Virapower lentiviral expression systems
Lentiviral systems for high level expression in dividing and non-dividing mammalian cell lines.
Cell culture and cell preparation
Heat Inactivating Serum
https://easyprotocols.com/showprotocol.php?prot=48
Cell Trypsinisation
https://easyprotocols.com/showprotocol.php?prot=52
FACS
FACS/Flow cytometry protocols
Flow cytometry is a powerful technique for analyzing large mixed populations of single cells. Fluorescence-activated cell sorting
(FACS) is a more complex system which not only quantifies the fluorescent signal but also separates the cells from a mixed population
that contains preselected characteristics (i.e. fluorescence intensity, size and viability).
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