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Western Blotting is an analytical technique used to detect specific proteins in a given sample. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane where they are detected using antibodies specific to the target protein.
Real-time PCR, also called quantitative real time PCR (Q-PCR/qPCR), is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.
Reverse Transcription PCR
Reverse transcription PCR includes two steps. The first step is reverse transcription, in which RNA is reverse transcribed to its DNA complement (complementary DNA, or cDNA) using reverse transcriptase and primers. The second step is amplification using traditional or real-time PCR.
Immunohistochemistry is a method of detecting the presence of specific proteins in cells or tissues. It is widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.
The indirect ELISA is used primarily to determine the strength and/or amount of antibody response in a sample. In the assay, the antigen of interested is immobilized by direct adsorption to the assay plate. Detection of the antigen can then be performed by using a matched set of primary antibody and conjugated secondary antibodies.
Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) is a technique to amplify few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. There are three major steps (Denaturation, Annealing and Extension) in a PCR and repeat for 30 or 40 cycles.
H&E stain is a popular staining method in histology. Its a combination of two dyes: the basic dye (hematoxylin) and the alcohol-based dye (eosin). In an H&E stain you will usually see both eosinophilia and basophilia: the nuclei of cells basophilic (blue), while eosinophilia is typical of cytoplasmic constituents (pink).
Cell Culture (Attached Cells)
Cells are cultured under précised conditions; A complete protocol on how to thaw, maintain and freeze attached cells is shown in this AbVideo.
Immunofluorescence is a technique to visualize a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye. We use the indirect immunofluorescence staining to perform cells fixed on slides and examine under a fluorescence microscope.
In competitive ELISA, unlabeled antibody is incubated in the presence of its antigen. Then these bound antibody/antigen complexes are then added to an antigen coated well. After washing, unbound antibodies are removed. The more analytes in the sample, the less antibodies will be able to bind to antigens in the well. The signal is then detected using labeled secondary antibodies and the decrease in signal is compared to a control. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.
The Sandwich ELISA measures the amount of analyte between capture antibody and detection antibody. The analyte needs to have two different epitope sites available for antibody binding.
Rotary Microtome Section
Rotary microtome is an instrument used to cut the blocks of tissue embedded in paraffin into micro fine slices (known as sections). Microtome is an important device in microscopy preparation, allowing for the preparation of samples for observation under transmitted light or electron radiation.
Cell Counting (Cell Suspension)
This AbVideo introduces the protocol of preparing suspension cell for counting. Hemocytometer or Cell Counting Chamber is frequently used to count cell numbers such as cell density of cultured cells in research labs and blood count and sperm count in clinical labs. For counting of attached cells, cells are trypsinized, mixed with a dye (Trypan Blue), loaded onto a hemocytometer and then counted under a microscope.
BLAST - PCR Primer Design
Primer-BLAST was developed to help users make primers that are specific to the input PCR template. It uses Primer3 to design PCR primers and then submits them to BLAST search against user-selected database. The blast results are then automatically analyzed to avoid primer pairs that can cause amplification of targets other than the input template.
Cre-Lox recombination is a special type of site-specific recombination. The Cre protein is a site-specific DNA recombinase. It can catalyze the recombination of DNA between specific sites in a DNA molecule. These sites, known as loxP sequences, contain specific binding sites for Cre that surround a directional core sequence where recombination can occur. It is often used in the generation of knockout and conditional knockout animals.
Protein Quantification (Bradford Assay)
Bradford assay is a rapid and accurate method to determine the concentration of protein. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. Within the linear range of the assay (~5-25 mg/ml), the more protein present, the more Coomassie binds.
Dot blot is a technique can be used as a semiqualitative method for rapid screening of a large number of samples. It is for detecting, analyzing, and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane.
Cell Counting (Attached Cells)
This episode of the cell counting series demonstrates the handling of attached cell before counting. Hemocytometer or Cell Counting Chamber is frequently used to count cell numbers such as cell density of cultured cells in research labs and blood count and sperm count in clinical labs. For counting of attached cells, cells are trypsinized, mixed with a dye (Trypan Blue), loaded onto a hemocytometer and then counted under a microscope.
A demonstration on the procedure of using MTT assay to assess the viability and the proliferation of regular cells with absorbance detection at 595nm is shown. This colorimetric assay measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase.
Plasmid DNA Extraction (Miniprep)
Miniprep plasmid DNA extraction kit is used when the starting E. coli culture volume is 1~5 ml of LB broth and the expected DNA yield is 20~30 μg. Other extraction and purification plasmid DNA Kits are available from varying manufacturers, named by size of bacterial culture, includes gigaprep, megaprep, and midiprep