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Does anyone know an effective buffer mix to use for high current Western transfers? We are successfully using the vendor's premixed buffer to transfer a wide range of protein sizes to PVDF membranes at 1A/25V for 10 min. We get great results with the vendor's expensive buffer.
I haven't been able to find a non-proprietary recipe that works. The best ones we have hold the 25V, but then have a decrease in conductivity (increase in resistance) where they will start at 1A, but drop to 0.4A by the end of the 10 min. The vendor's buffer seems to hold the high current and results in better transfers.
I don't know if it will be helpful to list all the variations I've tried in detail, but they have revolved around modifying a traditional Towbin buffer plus SDS, more glycine/Tris, or MgCl2. These were all various suggestions from people around the department, but I haven't found much published evidence for a buffer under these conditions.
I realize 1A (and no I don't mean 1mA) is a lot of current, but the vendor's system works really well. Any pointers on what I might try/add would be appreciated even if you don't have a worked out protocol.
Edit: Info from the MSDS indicates it has 3 reagents:
Listing of dangerous and non-hazardous components: Proprietary Reagent K 10-20% Proprietary Reagent EB II 5-10% Proprietary Reagent S 1.0-2.5% 7732-18-5 water 50-100% ·… Solvent content: Organic solvents: 0.0 % Water: 74.8 % Solids content: 25.2 %
[I'm not sure if this MSDS info should be put here, just because I'm looking for someone who has already used a high current buffer they know the formulation for, not a guess to what I have.]
So after a lot of work in optimization, I thought I would post what worked best for me. This buffer recipe was able to successfully transfer EGFR and insulin from the same lysate, and a clear band for both (large and small protein respectively). 10% SDS-PAGE gels were transferred at 1A for 10 min.
High Current Transfer Buffer
- 48 mM Tris
- 15 mM HEPPS
- 1.0 mM EDTA
- 1.3 mM NaHSO3
- 1.3 mM N,N-dimethylformamide
- 25 Mm gLY-GLY
- 20% Methanol (v/v)
I do hope this can help someone else, and want to site Garic et al as wonderful starting point. Their publication was made after I asked the question, as @user4148 pointed out.
The SB buffer has been around for quite a while, and I definitely have used it successfully before for both agarose and PAGE gel buffers for some time. The only thing is that you have to run the gel in a cold room (high voltage equals high temperature) and that you have to optimize the timing and conditions so that your samples don't run off the gel.
High Current (Speed) Transfer Buffer Recipe - Biology
WAYNE L. RICKOLL , . ELIZABETH S. HAYES , in Ecdysone , 1986
Gel electrophoresis (SDS–PAGE)
Aliquots of the NP-40 lysis buffer extracts were mixed with NP-40-urea lysis buffer with the ratio of protein to NP-40-urea lysis buffer kept constant for each sample (2 μg protein/μl buffer). Two-dimensional (2D) electrophoresis was carried out as described by O⟺rrell (1975) . Equal amounts of radioactivity (1 × 10 6 cpm) were loaded on all gels to be compared. The second SDS–PAGE dimension was run on a 7.5–17.5% acrylamide gradient slab gel, using a 4.5% acrylamide stacking gel.
NP-40 lysis buffer extracts were also analyzed on 7.5% SDS–PAGE. To compare each sample, aliquots of NP-40 lysis-buffer extracts containing equal amounts of radioactivity were added to SDS-sample buffer. The ratio of protein to SDS-sample buffer (2 μg/μl buffer) was kept constant for each sample. All gels were dried, and autoradiography was performed at −70°C using enhancing screens (Dupont Lightning Plus) and Kodak SB-5 X-ray film. At least three independent labelings and separations were carried out on both hormone-treated and non-treated cells.
The Human Immunodeficiency Virus (HIV)-derived lentivirus belongs to the Retroviridae family 1 . It is characterized by incorporating viral RNA into the DNA of dividing and non-dividing cells. To produce a lentivirus that is capable of infecting host cells, three types of vectors must be co-expressed in virus-producing cells (typically HEK293T cells): a backbone vector containing the transgene of interests and self-inactivating 3′-LTR regions, one construct expressing viral structure proteins and one vector encoding vesicular stomatitis virus glycoprotein (VSVG) for encapsulation 2 . The latest generation of the lentivirus system further separates the Rev gene from other structural genes, offering increased biosafety by reducing the possibility of reverse recombination 3 . Nowadays, lentivirus technology has become one of the most efficient tools to deliver exogenous genes into various types of cells both in vitro and in vivo 4,5,6,7 .
The purification of high-titer lentivirus (>10 7 TU/ml) from a large volume of virus-containing medium is crucial for the application of lentivirus. The routine concentration of the lentivirus requires ultracentrifugation with relative centrifugal force (RCF), typically exceeding 90,000 g 8,9,10,11,12,13 , to remove impurities and to ensure a high infection rate, especially for in vivo applications. This can be achieved only by an ultracentrifuge, which is not a common instrument in a lab. Moreover, the ability of low-speed centrifugation to produce a high-titer virus has not been thoroughly investigated. Thus, the relationship between RCF and the concentration efficiency of the lentivirus was systematically evaluated and the present data showed that sucrose gradient centrifugation with a relatively low speed (≤10,000 g) produces a high-titer virus that is comparable to a commercially available virus purified with ultracentrifugation in both yield and titer. Moreover, the optimal sucrose concentration is 10%. Thus, an efficient protocol for high-titer lentivirus purification that is easily achieved with a regular lab centrifuge is described.
Follow Western Blot wet transfer protocol
|1||Run the samples in SDS-PAGE as usual.|
|2||Prepare and mark the membrane (right size, PVDF or nitrocellulose membrane) with a pencil then soak it in Methanol for a few minutes, and rinse it with distilled water.|
|3||Equilibrate the membrane, pads, filter papers (4 pieces) and transfer foam in Transfer Buffer (store in 4°C) for 5 minutes.|
|4||Assemble transfer sandwich in the following order: black frame (negative electrode) >> foam >> 3 pieces of filter papers >> SDS-PAGE gel >> membrane >> 3 pieces of filter papers >> foam >> red frame (positive electrode). Make the marked side of membrane face the gel. Put it into transfer tank.|
Fig 2. Schematic picture of Western Blot wet transfer sandwich
|5||Transfer the gel at 80 V for 90 mins in cold room. Alternatively, use ice bag and magnet stirring bar to transfer the gel in room temperature.|
|6||Disassemble the gel pack, and use a pencil to mark the well imprints on the membrane.|
REAGENTS AND SOLUTIONS
½ MS medium plates
- 0.5× Murashige & Skoog medium including vitamins (Duchefa Biochemie, cat. no. M0255)
- 15 g/L sucrose
- Adjust pH to 5.7 with KOH
- 3 g/L Phytagel (Sigma, cat. no. P8169)
- Autoclave at 121°C for 15 min
- Cool to ∼60°C and pour into plates
- Store ≤6 months at 4°C
Coomassie Brilliant Blue R-250 destain solution
Coomassie Brilliant Blue R-250 stain solution
- 0.1% (w/v) Coomassie Brilliant Blue R-250
- 25% (v/v) isopropanol
- 10% (v/v) acetic acid
- Store ≤1 year at room temperature
Native protein extraction buffer
- 1× NativePAGE™ Sample Buffer (Thermo Fisher Scientific, cat. no. BN2008)
- 1× protease inhibitors (Roche, cat. no. 5056489001)
- 1% (w/v) n-dodecyl-β-D-maltoside (DDM Thermo Fisher Scientific, cat. no. BN2005)
- Prepare fresh immediately before use
Phosphate-buffered saline with Tween 20 (PBST)
- 1.8 mM KH2PO4
- 10 mM Na2HPO4
- 2.7 mM KCl
- 137 mM NaCl
- Adjust pH to 7.2 with 1 M NaOH
- Sterilize by autoclaving
- Store ≤6 months at room temperature
- Add 0.1% (v/v) Tween 20 immediately before use
Protein loading buffer, 4×
- 200 mM Tris·HCl, pH 6.8
- 8% (w/v) SDS
- 40% (v/v) glycerol
- 0.032% (w/v) bromophenol blue
- Store ≤1 year at room temperature
- Dilute to 1× and add 100 mM DTT before use
ALTERNATE PROTOCOL 1
ADDITIVE SCREEN TO OPTIMIZE PROTEIN STABILITY
This secondary protocol is ideal for screening a wide variety of additives once an optimized buffer has been identified in the Basic Protocol.
Same materials as for Basic Protocol 1
96-well deep well block containing 5× additive screen of choice (see Table 2 )
Composition of the 96-well additive screen
|A1||water||E1||5 mM EDTA|
|A2||water||E2||100 mM sodium fluoride|
|A3||water||E3||100 mM potassium fluoride|
|A4||water||E4||100 mM lithium chloride|
|A5||100 mM Urea||E5||100 mM potassium chloride|
|A6||250 mM Urea||E6||100 mM ammonium chloride|
|A7||500 mM Urea||E7||100 mM sodium iodide|
|A8||1 M Urea||E8||100 mM potassium iodide|
|A9||25 mM Guanidine HCl||E9||100 mM sodium bromide|
|A10||50 mM Guanidine HCl||E10||10 mM magnesium chloride|
|A11||100 mM Guanidine HCl||E11||10 mM calcium chloride|
|A12||250 mM Guanidine HCl||E12||5 mM manganese chloride|
|B1||500 mM Guanidine HCl||F1||5 mM nickel chloride|
|B2||1% (v/v) DMSO||F2||5 mM iron (III) chloride|
|B3||2% (v/v) DMSO||F3||5 mM zinc chloride|
|B4||2.5% (v/v) Glycerol||F4||5 mM cobalt chloride|
|B5||5% (v/v) Glycerol||F5||100 mM sodium formate|
|B6||10% (v/v) Glycerol||F6||100 mM sodium acetate|
|B7||15% (v/v) Glycerol||F7||100 mM sodium malonate|
|B8||20% (v/v) Glycerol||F8||100 mM sodium nitrate|
|B9||2.5% (v/v) D-Glucose||F9||100 mM sodium thiocyanate|
|B10||5% (v/v) D-Glucose||F10||100 mM sodium sulfate|
|B11||2.5% (v/v) Sucrose||F11||100 mM ammonium sulfate|
|B12||5% (v/v) Sucrose||F12||100 mM ammonium chloride|
|C1||2.5% (v/v) PEG400||G1||2 mM AMP + 5 mM MgCl2|
|C2||5% (v/v) PEG400||G2||2 mM ADP + 5 mM MgCl2|
|C3||2.5% (w/v) PEG1000||G3||2 mM ATP + 5 mM MgCl2|
|C4||5% (w/v) PEG1000||G4||2 mM AMPPNP + 5 mM MgCl2|
|C5||2.5% (w/v) PEG4000||G5||2 mM cAMP + 5 mM MgCl2|
|C6||5% (w/v) PEG4000||G6||2 mM GDP + 5 mM MgCl2|
|C7||2.5% (v/v) Ethylene Glycol||G7||2 mM GTP + 5 mM MgCl2|
|C8||5% (v/v) Ethylene Glycol||G8||2 mM cGMP + 5 mM MgCl2|
|C9||1 mM Octyl Glucoside||G9||2 mM NAD + 5 mM MgCl2|
|C10||2 mM CHAPS||G10||2 mM NADH + 5 mM MgCl2|
|C11||10 mM L-Proline||G11||10 mM Betaine|
|C12||50 mM L-Glycine||G12||1 mM Spermine|
|D1||25 mM L-Histidine||H1||1 mM Spermidine (add fresh each time)|
|D2||50 mM L-Arginine||H2||10 mM β-mercaptoethanol (add fresh each time)|
|D3||50 mM L-Glutamate||H3||5 mM DTT (add fresh each time)|
|D4||50 mM L-Arg/50 mM L-Glu||H4||2 mM TCEP (add fresh each time)|
|D5||25 mM L-Glutamine||H5||open for user-determined additives|
|D6||50 mM L-Lysine||H6||open for user-determined additives|
|D7||50 mM L-Cysteine||H7||open for user-determined additives|
|D8||50 mM Taurine||H8||open for user-determined additives|
|D9||50 mM Imidazole pH 7.6||H9||open for user-determined additives|
|D10||100 mM Imidazole pH 7.6||H10||open for user-determined additives|
|D11||250 mM Imidazole pH 7.6||H11||open for user-determined additives|
|D12||500 mM Imidazole pH 7.6||H12||open for user-determined additives|
Concentrations listed represent the final concentration in the thermal shift assay.
Stocks should be made at 5× concentration.
Note: Bring 5× additive screen up to room temperature from 4 ଌ storage before use ( Add enough of the optimized buffer (at 1× concentration, identified in the initial buffer optimization screen above) to protein stock in a 15 ml conical tube to obtain a final volume of 5 ml, sufficient to screen one 96-well assay plate. Add 4 µl of SYPRO Orange dye (stock concentration: 5000×). The final concentrations of protein and dye in the mixture should be 5 µM and 2×, respectively. Mix thoroughly by inverting the tube several times and place the solution in a multichannel pipette reservoir trough. Use a multichannel pipette to transfer 40 µl of solution into each well of the assay plate. Centrifuge room temperature 5× additive screen plate at 800 × g for 2 min at 25 ଌ. Carefully peel off adhesive aluminum sealing film. Use the multichannel pipette to add 10 µl of the 5× additive screen stocks from the 96-well deep well block to the assay plate. Mix the well content using the same pipette and tips by pipetting up and down several times. Design your 96-well additive screen so that there are at least four wells of no additives (i.e. water only in wells A1 in the 96-well additive screen plate) to assess the thermal shift of additives relative to the optimized buffer. Cover the assay plate with a sheet of optically clear adhesive and carefully seal each well. Reseal the additive screen with adhesive aluminum foil and store at 4 ଌ. Centrifuge the assay plate at 800 × g for 2 min at 25 ଌ to collect solutions in the bottom and remove bubbles from the wells. Place the assay plate into the real-time PCR instrument and start a temperature gradient program for protein thermal denaturation. Determine the thermal shift (ΔTm) of all conditions relative to the ‘no additive’ control to identify additives that promote stabilization of the protein. Equilibrate column with 5–10 column volumes of start buffer or until the baseline, eluent pH and conductivity are stable. Using prepacked columns is highly recommended to ensure the best performance and reproducible results. An evenly packed column ensures that component peaks are not unnecessarily broadened as sample passes down the column so that the best resolution can be achieved. Allow buffers, media or prepacked columns to reach the same temperature before use. Rapid changes in temperature, for example removing packed columns from a cold room and then applying buffer at room temperature, can cause air bubbles in the packing and affect the separation. Wash away storage solutions and preservatives before using any IEX medium. Increase the volumes used for column equilibration before the first run if using buffers containing detergents or a different counter-ion to the one in which the medium has been stored. Appendix 3 gives details on column packing. The volume required for the packed bed is determined by the amount of sample to be purified and the binding capacity of the medium. Pack a column that will have approximately 5-fold excess of the binding capacity required with a bed height up to 20 cm. Check column performance regularly by determining column efficiency and peak symmetry. Appendix 3. Note that this does not apply to HiTrap or HiPrep™ columns. (adapted from Kigawa et al. (2004), Preparation of Escherichia coli cell extract for highly productive cell-free protein expression, J. Struct. Fun. Gen., 5, 63-68) E. coli strain: BL21(DE3), BL21(DE3) CodonPlus RIL or variants (not pLysS!) ! around 7 ml of S30-ribosome-lysate can be isolated from 1 litre of culture ! Per litre medium:
Column and Media Preparation
Protocol for S30-ribosome-lysate
5.6 g KH2PO4 28.9 g K2HPO4 10 g yeast extract 15 mg Thiamine add after sterilisation (filtered sterile) 40 ml 25% Glucose add after sterilisation (filtered sterile)
Add enough of the optimized buffer (at 1× concentration, identified in the initial buffer optimization screen above) to protein stock in a 15 ml conical tube to obtain a final volume of 5 ml, sufficient to screen one 96-well assay plate. Add 4 µl of SYPRO Orange dye (stock concentration: 5000×). The final concentrations of protein and dye in the mixture should be 5 µM and 2×, respectively.
Mix thoroughly by inverting the tube several times and place the solution in a multichannel pipette reservoir trough. Use a multichannel pipette to transfer 40 µl of solution into each well of the assay plate.
Centrifuge room temperature 5× additive screen plate at 800 × g for 2 min at 25 ଌ.
Carefully peel off adhesive aluminum sealing film. Use the multichannel pipette to add 10 µl of the 5× additive screen stocks from the 96-well deep well block to the assay plate. Mix the well content using the same pipette and tips by pipetting up and down several times.
Design your 96-well additive screen so that there are at least four wells of no additives (i.e. water only in wells A1 in the 96-well additive screen plate) to assess the thermal shift of additives relative to the optimized buffer.
Cover the assay plate with a sheet of optically clear adhesive and carefully seal each well. Reseal the additive screen with adhesive aluminum foil and store at 4 ଌ.
Centrifuge the assay plate at 800 × g for 2 min at 25 ଌ to collect solutions in the bottom and remove bubbles from the wells.
Place the assay plate into the real-time PCR instrument and start a temperature gradient program for protein thermal denaturation.
Determine the thermal shift (ΔTm) of all conditions relative to the ‘no additive’ control to identify additives that promote stabilization of the protein.
Equilibrate column with 5–10 column volumes of start buffer or until the baseline, eluent pH and conductivity are stable.
Using prepacked columns is highly recommended to ensure the best performance and reproducible results. An evenly packed column ensures that component peaks are not unnecessarily broadened as sample passes down the column so that the best resolution can be achieved.
Allow buffers, media or prepacked columns to reach the same temperature before use. Rapid changes in temperature, for example removing packed columns from a cold room and then applying buffer at room temperature, can cause air bubbles in the packing and affect the separation.
Wash away storage solutions and preservatives before using any IEX medium.
Increase the volumes used for column equilibration before the first run if using buffers containing detergents or a different counter-ion to the one in which the medium has been stored.
Appendix 3 gives details on column packing. The volume required for the packed bed is determined by the amount of sample to be purified and the binding capacity of the medium. Pack a column that will have approximately 5-fold excess of the binding capacity required with a bed height up to 20 cm.
Check column performance regularly by determining column efficiency and peak symmetry. Appendix 3. Note that this does not apply to HiTrap or HiPrep™ columns.
(adapted from Kigawa et al. (2004), Preparation of Escherichia coli cell extract for highly productive cell-free protein expression, J. Struct. Fun. Gen., 5, 63-68)
E. coli strain: BL21(DE3), BL21(DE3) CodonPlus RIL or variants (not pLysS!)
! around 7 ml of S30-ribosome-lysate can be isolated from 1 litre of culture !
Per litre medium:
All buffers and stock solutions should be prepared with diethylpyrocarbonate (DEPC)-treated H2O (DNAse/RNAse-free).
All stock-solution can be stored @ -20°C for weeks except creatine kinase. Creatine kinase has to be dissolved in 30 mM Glycin, pH = 9.0 + 20 mM DTT. After freezing in liquid nitrogen, it is stable @ -80°C for weeks.
All individual amino acids have to be dissolved as described on package. The mixture contains 5 mM of each amino acid with a pH of 7.5 adjusted with KOH.
The procedure is suitable for all types of tissues from a wide variety of animal, blood, plant species and soil.
The following protocol is designed for small and large tissue samples (tissue volume 100-200 μl).
Note that isolating genomic DNA not requires gentle mixing because the DNA not be sheared by vortexing.
Kalendar R, Boronnikova S, Seppänen M 2021. Isolation and purification of DNA from complicated biological samples. Methods in molecular biology, 2222: 57-67. DOI:10.1007/978-1-0716-0997-2_3
CTAB protocol for the isolation of DNA
- CTAB solution: 2% CTAB, 1.5 M NaCl, 10 mM Na3EDTA, 0.1 M HEPES-acid
100 ml: 2 g CTAB, 2.4 g HEPES-acid, 2 ml 0.5 M Na3EDTA, 30 ml 5 M NaCl
- Chloroform-isoamyl alcohol mix (24:1)
- 100% isopropanol (isopropyl alcohol, 2-propanol)
- 70% ethanol
- 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0)
- 2 ml Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball (6 mm), grind in the MM300 Mixer Mill for 2-10 min at 30 Hz.
- In 2 ml tube with mechanically disrupted seeds/leaves/herbarium or DNA solution (CTAB purification) add 1 ml CTAB solution buffer, mix in the MM300 Mixer Mill for 2 min at 30 Hz and incubate the samples at 65°C during 1 hour.
- Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the clarified solution to a new 2 ml microcentrifuge tube contains an equal volume of chloroform.
- Mix very well for 3 minutes in the MM300 Mixer Mill at 30 Hz.
- Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube which contains 600 μl 2-propanol, vortex well and centrifuge the tubes at maximum speed in a microcentrifuge for 2 minutes.
- Discard supernatant and wash pellet by adding 1.8 ml 70% EtOH, vortex very well. Centrifuge at maximum speed for 2 min and discard ethanol.
- The DNA pellet do not dry and dissolved immediately in 300 &mul 1xTE, pH 8.0 at 55°C for 5-10 minutes.
CTAB protocol for the isolation of DNA from difficult tissues (high levels of secondary metabolites or polysaccharides), herbarium and soil
- CTAB solution: 3% CTAB, 1.5 M NaCl, 10 mM Na3EDTA, 0.1 M MOPS-acid
100 ml: 3 g CTAB, 2.1 g MOPS-acid, 2 ml 0.5 M Na3EDTA, 30 ml 5 M NaCl
- Mini spin column: HiBind® (Omega Bio-tek) or NucleoSpin® (MN) or compatible with the following Qiagen kits: Qiaprep Spin Mini, QiaQuick Gel Extraction, QiaQuick PCR Clean-up, DNeasy Plant DNA
- Buffer QG (5.5 M guanidine thiocyanate, 20 mM Tris-HCl pH 6.6)
- DNA Wash Buffer PE (80% ethanol, 20 mM NaCl, 2 mM Tris-HCl, pH 7.5)
- 96% ethanol
- Elution buffer (0.5 mM EDTA, 10 mM Tris-HCl, pH 9.0)
- 2 ml Eppendorf Safe-Lock microcentrifuge tube with herbarium sample (the sample mass should not exceed 50 mg) and glass ball (6 mm) grind in the MM300 Mixer Mill for 15 min at 30 Hz.
- Add 1 ml CTAB solution buffer, vortex to mix thoroughly and incubate the samples at 65°C during 1-2 or more hours.
- Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the clarified solution to a new 2 ml microcentrifuge tube contain an equal volume of 96% ethanol (50-100% of the CTAB solution volume). Vortex to mix thoroughly.
- Insert a HiBind DNA Mini column into a 2 ml collection tube. Transfer up to 700 μl sample from step 3 to the HiBind DNA Mini column. Centrifuge at maximum speed for 30 sec at room temperature.
- Using the same collection tube, repeat previous step until all the CTAB mix solution has passed through the HiBind DNA Mini column. Centrifuge at maximum speed for 30 sec. Discard the filtrate and collection tube.
- Add 500 μl 96% ethanol. Centrifuge at maximum speed for 30 sec. Discard the filtrate and reuse collection tube.
- Add 500 μl QG buffer. Centrifuge at maximum speed for 30 sec. Discard the filtrate and reuse collection tube.
- Add 700 μl DNA wash buffer PE. Centrifuge at maximum speed for 30 sec. Discard the filtrate and reuse collection tube.
- Using the same collection tube, repeat previous step for second DNA wash buffer wash step. Centrifuge at maximum speed for 1 minute.
- Transfer the HiBind DNA Mini column into a clean 1.5 ml tube. Add 200 μl elution buffer and heat at 55°C for 10 minutes.
- Centrifuge at maximum speed for 1 minute. Store eluted DNA at -20°C.
Proteinase K method for DNA extraction protocol
- In 2 ml Eppendorf Safe-Lock tube with mechanically disrupted animal or plant tissues add fresh 500 μl of extraction buffer (0.8 M guanidine thiocyanate, 10 mM EDTA, 5% Tween 20, 0.5% Triton X-100, 50 mM HEPES-acid*) with 200 &mug of proteinase K, vortex very well and incubate the samples at 55°C for several hours or better overnight at 37°-55°C (the longer the better, until dissolve tissue) with occasional vortexing.
- Add 700 μl of chloroform, vortex very well for 1 minute creating an emulsion (in the MM300 Mixer Mill at 30 Hz) optionally: incubate the samples at 55°C during 30 min.
- Spin at maximum speed in a microcentrifuge for 5 minutes.
- Transfer the supernatant into a new 2 ml tube containing 500 μl of 2-propanol and 100 &mul 3M Na-acetate, vortex very well, and centrifuge the tubes at maximum speed in a microcentrifuge for 4 minutes.
- Discard the supernatant and add 1.8 ml of 70% ethanol into tube and vortex well centrifuge the tube for 5 minutes at 14000 rpm and again discard the supernatant.
- Do not dry DNA pellet and dissolved immediately in 300 μl of 1xTE, pH 8.0 (with RNAse A) at 55°C for 10-20 minutes.
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8 Top Tips For Immunoprecipitation
I mmunoprecipitation, or IP, is a well-established technique for the isolation of proteins from, well, a rather chaotic mixture of many other proteins in solution. The solution could be anything from a tissue homogenate to a cell lysate, but the goal remains the same: isolation of one specific protein and nothing else – or at least very little else (as in the case of co-immunoprecipitation). There are even protocols adapted from the protein IP procedure for the isolation of chromatin (ChIP) and RNA (RIP), but in this blog post we will only focus on the protein version and provide tips on how to perform better IP experiments.
1. Lysate Preparation
There’s an old adage in the English language: You only get out what you put in, and this saying is true of IP experiments. You want to start with a fair amount of material aim for between 1 and 3 mg of total protein for every 0.2-0.5 ml of your starting sample volume. You should also aim to keep your target protein as happy as possible throughout the disruptive procedure of cell or tissue lysis. It’s a balancing act really: the ideal lysis buffer will leave proteins in their native conformations and minimize the denaturation of antibody binding sites, but also release enough protein for you to capture and analyse. The Proteintech lab usually uses RIPA buffer, which enables efficient cell lysis and protein solubilization, while avoiding protein degradation and interference with the proteins’ immunoreactivity and biological activity. Using RIPA buffer results in low background in IP and is particularly useful for nuclear membrane disruption for nuclear extracts, but it can denature kinases, so watch out if kinase activity is your goal! And don’t forget to add a protease inhibitor (and a phopshatase inhibitor if you’re looking at phosphorylation!)
You can find our RIPA buffer recipe here . If you’re looking for an alternative to RIPA buffer, our main advice is to go easy on the salt and detergents if you can! Stick to tried and tested buffers (you can find may recipes on scientific forums) and if you do need to tweak your buffer recipes a little, stick within the ranges below recommended by Harlow and Lane, page 231 (see the box below).
2. Choose your antibody wisely
In general, you should consider using a polyclonal antibody, where possible, for the capture of your target protein. Figure 1 provides a good visual explanation as to why this is the case, but in general you’re aiming to establish an antigen-antibody immune complex at the start of the IP procedure. As polyclonals bind multiple epitopes on the target protein, they retain a greater amount of it. As the goal is to wash any unwanted proteins and constituents away later, retention rates need to be pretty high so as not to wash your sample away too! Therefore a good-quality polyclonal should be your first choice for an IP procedure.
Figure 1: Polyclonal vs Monoclonal as capture antibodies. Polyclonals form tighter binding immune complexes with higher retention rates.
Proteintech’s vast range of polyclonals are ideal for IP as, not only do they fit the job description above, they are also good at recognizing native, intact epitopes. Native epitopes will still retain their three-dimensional confirmations, and as Proteintech antibodies are raised against whole proteins they do the job of recognizing whole, intact proteins in the IP sample rather well. (That being said, it is a good idea to use a monoclonal antibody for detection at the Western blotting (WB) stage, but we’ll get to that later!)
3. To preclear or not to preclear
Preclearing is a step that prevents any non-specific proteins, lipids, carbohydrates or nucleic acids that you don’t want from binding to the solid support you are using in your IP experiment. The step is performed by incubation of the lysate with the solid support (e.g. agarose beads) in the absence of the capture antibody. The solid support used for preclearing, and therefore anything bound to it, is then discarded. This step is dependent on the type of solid support. It also comes down to how good your detection antibody is and how much of your protein of interest might be in the lysate. Some solid supports are more likely to bind non-specifically to certain proteins, so check the manufacturer’s recommendations first , you might be able to skip preclearing all together.
There’s also a preclearing technique that involves the addition of a non-specific antibody of the same isotype as the capture antibody –this can be done to remove anything that might also bind non-specifically to the capture antibody during precipitation.
In general, we do not preclear lysates in the Proteintech lab when performing our IP validations at all for the reason that preclearing is difficult to control when you’re performing a large number of IPs on a regular basis (see also the part about quality of the detection antibody above). It’s not always necessary, and requires careful assessment of the amount of preclearing reagents required to avoid unecessary sample loss.
4. Washing and eluting – mind how you go!
The effect of centrifugation on the outcome of an IP experiment is often underestimated. Centrifugation of the immune complex at too high a speed will disrupt the antibody-antigen interactions in the immune complex and cause sample loss before elution. For that reason we recommend that centrifugation be omitted from the washing and elution steps entirely. Instead, perform washing and obtain elutions using gravitational flow through filter columns loaded in microfuge tubes.
5. Eluted samples vs boiled samples
Traditional measures to improve signal-to-noise ratios aim to retain as much target protein as possible to the detriment of efficient immune complex separation in the final stages of IP. Steps taken toward this end usually involve the omission of reducing agents from the elution buffer or the boiling step prior to SDS-PAGE. Sometimes elution is omitted entirely, and samples still attached to the solid phase are boiled following the washing stages (see Figure 2A ). The results are WBs that may have improved target signal, but huge amounts of non-specific staining contributed by the presence of the solid phase (see Figure 2 ). We strongly recommend that you don’t do this, and elute your samples from the solid support first before gel loading.
Figure 2: Shows comparison of boiling samples (still attached to solid support) immediately after washing (A) and after elution (B) using PH 2 buffer (150 mM glycine, 500 mM NaCl) and neutralized before loading on an SDS-PAGE gel. Capture and detection antibody was anti-NFKBIA (cat. no. 10268-1-P) secondary detection was done using HRP-conjugated Protein A (see the section on alternative detection methods). Cell lysate: HeLa.
6. Keep your fractions!
It’s advisable to hang on to all of your IP fractions (including samples from any preclearing you perform) to anaylze by SDS-PAGE later, should your initial attempts at immunopreciptation be unsuccessful.
7. Antibody pairs
The use of antibody pairs, i.e. a capture antibody from one species, and an antibody for WB detection from another, is an additional factor to consider when planning an IP experiment. The antibody selection process involved should ensure both antibodies recognize different epitopes of the target protein, in addition to originating from different species. The antibody type (i.e. polyclonal or monoclonal) is also important. We suggest a combination of a polyclonal capture antibody and a monoclonal antibody for detection where possible this ensures maximum capture efficiency with high detection specificity. See the example in Figure 3 .
Figure 3: Comparison of using a polyclonal capture antibody (10782-2-AP) and a monoclonal detection antibody (60019-1-Ig) (A) with the same antibody (10782-2-AP) for both capture and detection (B). Secondary detection was performed using HRP-goat anti-mouse (A) and HRP-goat anti-rabbit (B). Cell lysate used was K-562 whole cell lysate.
8. Try an alternative detection method!
Anyone who’s ever done an IP experiment before will know that the heavy chain (HC) and light chain (LC) of the capture antibody can interfere with your target signal. This is an unavoidable artifact of having to use the same antibody for capture and detection, and then use a species specific antibody on top of that for secondary detection.
Harlow, Ed, and David Lane. Using Antibodies. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1999.
Bonifacino, Juan S. et al. Current Protocols in Immunology 8.3.1-8.3.28, New York: John Wiley, 2001.