Table 2.

Different western blot methods to transfer proteins from gel to membrane

Methods	Brief Description	Publication s using this method *	Notes	References
Vacuum blotting	This technique is based on a suction force of a pump to transfer proteins from a gel to a nitrocellulose membrane connected to a slab dryer system.	[100–104]	Efficiently transferred hemolymph proteins and homogenates.	[4,105]
Centrifuge blotting	Protein is eluted and transferred to PVDF membrane by centrifugation instead of electro blotting. SDS-PAGE gel with protein of interest was immersed in 1M KCl for 2 min. The clear protein bands were then cut and centrifuged at 3000 × g for 1 hr along with PVDF and dialysis membranes after soaking them in the eluent.	[106,107]	Dialysis membrane was used to retain non- immobilized proteins which was placed below PVDF membrane	[4]
Multiple tissue blotting	Premade human multiple tissues western (MTW) blots allow detection of tissue-specific protein expression. Whole human tissues proteins are extracted, fractionated by SDS-PAGE gels, transferred to PVDF membranes and are incubated with antibodies specific to target protein.	[108]	Allows western blotting of human tissues by laboratories that do not usually have access to this tissue.	[109]
Electroblotting of proteins to Teflon tape and membranes for N and C terminal sequence analysis	Proteins are electro transfer to low-density Teflon tape and GORE-TEX expanded polytetrafluorethylene membranes. These membranes were soaked with absolute ethanol before electrotransfer. Following transfer blots were stained with 0.005 % sulforhodamine B in 30 % methanol for 10 min to visualize proteins.	[110]	Teflon blots were found to be suitable for amino acid analysis, in situ proteolytic digestion, N- and C- terminal sequencing and are inert to the chemistry used on C-terminal sequencers.	[4]
Two-step transfer of low and high molecular -weight proteins	This is a two-step method for transfer of high- molecular-weight (greater than 400,000) and low molecular-weight (less than 20,000) proteins to nitrocellulose membranes from polyacrylamide gels. In first step low-molecular-weight proteins are eluted for 1 hr at a low current density. Followed by the second step of prolonged electro-transfer at high current density for 16–20 hr. To enhance protein transfer SDS at a 0.01 % concentration was added to transfer buffer. In addition, after electrotransfer the nitrocellulose membrane was air-dried to minimize protein loss during subsequent processing.	[111–114]	This method is suitable for all kind of polyacrylamide gel systems as well as for proteins obtained from various cell types.	[4,109,111]
Transfer of high and low molecular weight proteins using heat	The high and low molecular weight proteins are efficiently transferred to nitrocellulose membrane with the use of normal transfer buffer that was heated to 70–75 °C. The electrotransfer was done for 10 min/ 7% SDS-PAGE (0.75mm) gels, 15 min/10% and 12.5% gels (0.75mm) gels and 20 min/7%, 10% and 12.5% (1.5 mm).	[26,115,116]	Rapid and efficiently transfer both low and high molecular weight proteins. Heating increased the gel permeability allowing easy transfer of proteins trapped in gel matrix.	[26]
Semi-dry Electroblotting of peptides and proteins from acid-urea polyacrylamide gels	Used low pH PAGE system by adding acidic urea to resolve various proteins and peptides that otherwise would not able to be resolved by SDS-PAGE. Proteins are transferred to PVDF membranes at 115 mA and 5 V for 15 min in a transfer solution that contains 5 % acetic acid.	[117]	Faster and convenient method used which can be used for DNA and RNA electroblotting as well.	[4]
Transfer of silver- stained proteins from polyacrylamide gels to PVDF membranes	Silver-stained proteins are efficiently transferred to PVDF from a polyacrylamide gel by rinsing the gel in 2× SDS Laemmli buffer prior to transfer.	[118,119]	Some silver stained proteins were found to be transferred directly without the rinsing of gel in Laemmli buffer.	[4,120]
Enhanced protein recovery using square wave alternating voltage after electro transfer	In this method, the combination of square wave alternative voltage (SWAV) and CAPS buffer has been used to transfer protein from gels previously soaked in deionized water and equilibrated two times in the cathodic blotting buffer for 5 min.	[121]	Compared to standard blotting technique 65 % more protein was recovered using this technique.	[4,122]
PEG-mediated immunoblotting transfer	Proteins separated on SDS-PAGE were soaked for 2 hr in a 30 % PEG 2000 buffer to fix the proteins. This was followed by electrotransfer of proteins from the gel to PVDF membrane for 24 –48 h at 200 mA/120 V in a −20 °C freezer.	[123,124]	PEG 1000, 1500 & 2000 increased the sensitivity of immunoblotting by 10–100 fold. However, PEG less than 1000 had no have remarkable effect.	[4,125]
Acid electroblotting on activated glass	Proteins were electrotransferred to activated glass instead of nitrocellulose or PVDF to obtain sub picomolar levels of proteins. The activated glass fiber paper support was found to be extremely stable for sequencing conditions. To activate glass various treatments were employed: (a) Acid Etching: Whatman GF/C or GF/F sheets were treated with TFA in a Petri dish for 1 h at room temperature and then dried completely. TFA treatment can adsorb about 7—10 μg/cm 2 of proteins. (b) Acid Blotting: After SDS PAGE the gels were incubated in 0.5 % acetic acid containing 0.5 % NP-40 for 10 min at room temperature. At low pH, the positive charged proteins were electro transferred to glass fiber paper.	[23,126]	Both nitrocellulose and nylon membranes are not suitable for sequencing chemistry. The underlying mechanism for protein adsorption on activated glass fiber was due to the ionic interaction of the positively charged proteins with the negative charges on the glass fiber sheet.	[4,23]
Wet transblot	In this method, the nitrocellulose or PVDF membrane is sandwiched between the gel, filter papers and a support pad facing anode. The transfer sandwich is secured in a cassette and is submerged inside a Trans-blot buffer tank with platinum wire electrodes and filled with cold transfer buffer. Finally, electrotransfer is carried out to transfer proteins from gel to membrane.	>1000	Good transfer efficiency, but requires large volume of transfer buffer with methanol. Transfer is carried out by placing an ice pack within the transfer tank or by placing the whole apparatus in a cold room.	[109]
Fast transfer (Rapid electroblotting)	This is a recent technique developed by Thermo Scientific for rapid transfer of proteins from gel to membrane using the Pierce™ G2 Fast Blotter. The technique utilizes the combination of high-ionic strength transfer buffer and a high current power supply (10 fold or more) that increases the transfer efficiency like wet or semidry transfer and can be achieved in 5–10 min.	None	Efficiently transfer of low, medium and high molecular weight proteins in just 10 min.	[127]
Trans-Blot Turbo Transfer	Highly efficient and rapid method for protein transfer that allows transfer of protein in just 3 min. The Trans-Blot Turbo Transfer utilizes an optimized buffer, membrane, filter paper and the Trans-Blot Turbo blotting apparatus.	>200	Transfer mini gels in 3 min Higher transfer efficiency and high throughput	[128]

^*other than the original paper describing the method. Abbreviations: polyvinylidene difluoride (PVDF); 3-(cyclohexylamino)-1-propane- sulfonique acid (CAPS); polyethylene glycol (PEG); trifluoracetic acid (TFA); Nonidet P-40 (NP-40); matrix-assisted laser desorption/ionization (MALDI); enzyme-linked immunosorbent assay (ELISA); hINF (human interferon)-γ.