![]() ![]() The sequence of adding specimens or controls first is flexible. Add 20 µl each of specimens to their respective wells, and followed by 20 ul each of Strong Reactive, Weak Reactive, and Non-Reactive Controls to their respective wells.Failure may result in watery marks on developed strips for some specimens.) Incubate the strips for 1 to 2 minutes at room temperature on a rocking platform (speed of 12 to 16 cycles per minute).Include strips for Strong Reactive, Weak Reactive, and Non-Reactive controls. Using forceps, carefully remove the required number of STRIPS from the tube and place numbered side up into each well.Observe the bands (See Interpretation of Results) and grade the results. Do not apply adhesive tape over the developed bands. Mount strips on the worksheet (non-absorbent white paper).Allow strips to dry in the wells of the tray.Aspirate the substrate and rinse the strips at least three times with reagent grade water to stop the reaction (A dark background can result if washing is.(Note: The reaction can be stopped before 15 minutes if all the bands are visible for high anti-HIV titer samples to avoid over development of bands and Cover the tray and incubate for 15 minutes on the rocking platform.Add 2 ml of substrate solution to each well.Cover the tray and incubate for 1 hour at room temperature on the rocking platform.Add 2 ml of the working conjugate solution to each well.(Note: Each wash cycle consists of dispensing 2 ml of diluted wash buffer, soaking time of 5 minutes, and aspiration.) Wash each strip 3 times with 5 minutes soak on the rocking platform between each wash. Change aspirator tips between samples to avoid cross-contamination. Tilt the tray to aspirate the mixture from the wells. Carefully uncover the tray to avoid splashing or mixing of samples.Cover the tray with the cover provided and incubate for 1 hour at room temperature on the rocking platform. ![]() Add 20 µl each of specimens to their respective wells, and followed by 20 µl each of Strong Reactive, Weak Reactive, and Non-Reactive Controls to their respective wells.Tilt the tray slightly and add the specimen(s) where the buffer is collected at the lower end of each well as per the next step. Add 2 ml of blotting buffer to each well.Incubate the strips for 1 to 2 minutes at room temperature (25 ± 3☌) on a rocking platform (speed of 12 to 16 cycles per minute).Using forceps, carefully remove a required number of strips from the tube and place numbered sides up into each well.Add 2 ml of diluted wash buffer to each well.Overnight Assay ( long procedure) Rapid Assay Procedure Test procedure according to the manual of MP Diagnostics for HIV BLOT 2.2 It can be further completed by either chromogenic detection or.Protein transfer to a membrane: membrane stain and blocking and washing.Gel electrophoresis of protein: It may be performed by gel stain or.Sample preparation ( protein extraction).Paper towel, adhesive tape, worksheet (non-absorbent white paper).Sodium hypochlorite for decontamination.Aspirator with sodium hypochlorite trap.Precision Pipettes ranging from 20µl to 1000µl dispensing volume.Rocking platform (designed with a rocking speed range of 12 to 16Ĭycles per minute, and which moves through a 5° to 10° tilt to wash.Incorporated with HIV-1 viral lysate, a.The sodium dodecyl sulfate (SDS) Polyacrylamide gel electrophoresis (PAGE) technique is a prerequisite for Western blotting. A technique for detecting specific proteins separated by electrophoresis by the use of labeled antibodies. In Western blotting, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein. It is an Immunoblotting technique that relies on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules. Western blot: It is applicable for transferring protein andĮastern blot: It is a biochemical technique used to analyze protein post-translational modifications (PTM) and is most often used to detect carbohydrate epitope. Northern blot: It uses for transferring RNA. Southern blot: It uses for transferring DNA. Blot is a technique for transferring DNA, RNA, and proteins onto a carrier so they can be separated, and often follows the use of gel electrophoresis. It is also called immunoblotting and it is a technique for detecting specific proteins separated by electrophoresis by the use of labeled antibodies. Western blot test is a confirmatory test of HIV-AIDS and it is positive as shown above image. ![]()
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