From: Emerging techniques of western blotting for purification and analysis of protein
Methodology and reference | Description | Merits of methods | References |
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Transfer by trans blot turbo | Extremely quick and efficient protein transmission technique that enables protein transport in only 3Â min. It utilizes the apparatus of blotting, filter paper, membrane, and buffer (optimized) | Three minutes higher effectiveness and high flow rates for mini gel transfer | [16] |
Electroblotting (rapid) | This is a recently designed methodology for quick protein transmission from gel to membrane with the blotter (Pierce G2 quick) from ThermoScientific. The method incorporates high-ionic transmission buffers with an elevated power source (tenfold or even more), which enhances transmission efficiency, such as semi-dry and wet transmission, and it can be accomplished in about 5–10 min | Efficient transmit, in about ten min, of proteins (high, medium, and low molecular weight) | [17] |
Transblot (wet) [18] | In this technique, a pad of support, filter paper, and gel are utilized to the sandwich membrane of PVDF or nitrocellulose. The transmission sandwich is safeguarded in a cassette, immersed in a buffer of trans-blot with electrodes of platinum wiring and cold transmission buffered. Eventually, electron transmission of proteins to the membrane from the gel is performed | The great effectiveness of transmission, but takes a significant amount of methanol buffer solution. The transmission is done by placing a cold pack inside or by putting the entire device in a deep freezer | Â |
For two h in a buffer of 30% PEG 2000, proteins segregated by SDS-PAGE were drenched to resolve the proteins. Electric transmission of proteins in a − 20 °C refrigerator to the membrane of PVDF from the gel was accompanied by 24–48 h at 200 mA/120 Volt | PEG 2000,1500 and 1000 improved the blotting sensitivity by ten- hundred times |  | |
Electro-transfer (voltage alternated by square wave) [22, 23] | This technique is being used to transmit gel protein, initially drenched in deionized water and balanced in the cathodium-blotting buffer 2 times in 5Â min by CAPS and SWAV (square-wave alternative voltage) | 65 percent more protein has been managed to recover utilizing this method than the conventional blotting methodology | Â |
Transmission of protein stained with silver to membrane of PVDF from gel of polyacrylamide | Protein stained with silver is transferred effectively from the gel of polyacrylamide to PVDF by soaking the gel in buffer (Laemmli SDS) before transmission | Some proteins have been directly transferred to Laemmli buffer without gel rinsing | |
Protein and peptide electroblotting (semi-dry) from gels of polyacrylamide (acid-urea) | System of PAGE (low pH) was utilized by addition of urea (acidic) to resolve protein molecules alternatively not addressed by SDS-PAGE. A transmission solution containing five percent acetic acid will transmit proteins to membranes of PVDF at 5Â V and 115Â mA for 15Â min | Quicker and more convenient methodology and utilized for electroblotting of DNA and RNA too | [27] |
Heat transmission of proteins | With the standard transmission buffer exposed to heating to 75 °C, the lower and higher molecular mass proteins are effectively transmitted to the membrane of nitrocellulose | Heat treatment enhances the gel permeation, making it much easier to transmit protein locked into the matrix of gel | |
Analysis of the sequence of proteins (N and C terminal) by electroblotting to membranes and tape of Teflon | Proteins are transferred (electro) to the membrane of polytetrafluoroethylene (GORE-TEX) and tape of Teflon. Before actually electrotransfer, such membranes have been drenched with 95% ethanol. After transmission blots, 0.005% sulforhodamine B was stained to visualise proteins in 30% methanol over 10Â min | The blots of Teflon, which are inert to the C-terminal sequencing, are considered as appropriate for the assessment of amino acids, digestion of proteolytic in situ, and sequencing of N and C terminal | [30] |
Blotting of multiple tissues | Numerous blots of Western tissue of human premade enable the identification of specific tissue of expression of the protein. Proteins from all of the skin cells, split into membranes of PVDF and incubated with target-protein specific antigens, are processed by gels of SDS-PAGE | Permits western blot analysis by labs with no availability to the tissue in body cells | |
Blotting by vacuum | This method is used to extract proteins from the gel into the membrane of nitrocellulose attached to the dryer device by a pump suction power | Homogenates and proteins of haemolymph are transferred efficiently |