ELISA/IHC/IF/WB/IP/ChIP
Enzyme-linked immunosorbent assay (ELISA) is widely used in immunology to detect the presence of proteins or other antibodies in a sample. For instance, it is used as an initial detection tool for HIV, based on the interaction of an antibody with antigen presented by the virus. There are several different ELISA methods: indirect ELISA, sandwich ELISA and competitive ELISA are the most commonly applied. All three methods have similar steps:
1) Attach antigens or primary antibodies or their complexes to a solid surface.
2) Wash away unbound substances.
3) Block exposed sites on the solid surface.
4) Add detection antibodies and/or enzyme-conjugated secondary antibodies.
5) Develop color by adding substrates that react with the enzymes.
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For a detailed step-by-step guide to Indirect ELISA download the pdf protocol below.
Immunohistochemistry (IHC) allows you to visualize proteins in tissue while remaining its microstructure. It helps to demonstrate the exact position and distribution of the protein of interest in the analyzed tissue section. The advantage of this visualization is that it allows for comparison between, for example, healthy and diseased tissues. Briefly, in an IHC experiment, the antigen of interest is localized by the binding of an antibody. The antibody-antigen interaction is then further visualized via chromogenic or fluorescent detection.
The IHC protocol contains many steps that may require optimization to ensure specific antibody binding and optimal visualization of the target protein. As the IHC protocol contains many different variable factors, it can be challenging to find the best working conditions to obtain strong and specific staining.?
All steps in the following protocol are carried out at room temperature unless stated otherwise. ?Recipes for all solutions highlighted ?bold ?are included at the end of the protocol.
Immerse slides in xylene for 10 minutes. Repeat this step again in fresh xylene ?for 10 minutes. (If required, repeat a third time in fresh xylene for another 10 minutes.)
Rehydrate sections by sequentially incubating with 100%, 95%, 80% and 60% ?ethanol for 5 minutes each.
Rinse sections with distilled water three times for 3 minutes each.
Transfer slides to a microwave-safe container and cover with ?Citrate buffer? or Tris-EDTA (TE) buffer.
Heat in the microwave on medium power for 10 minutes.
Allow slides to cool in the Citrate buffer or Tris-EDTA (TE) buffer ?for approximately ?35 minutes.
Rinse slides three times with 1x?TBST? for 3 minutes each.
Incubate slides with 3% H2O2 ?solution (diluted in distilled water) for 10 minutes ?to quench endogenous peroxidase activity.
Rinse slides three times with 1x?TBST ?for 3 minutes each, then rinse slides three ?times with distilled water for 3 minutes each.
Prepare 5% normal blocking serum in 1x?TBST. The serum should be derived from ?the same species in which the secondary antibody was raised. Block the sections ?for 1 hour. (Alternatively, use 5% BSA in 1x?TBST ?for blocking if the corresponding ?serum is not available.)
Incubate sections with primary antibody diluted in 1x?TBST ?for 1 hour, or ?overnight at 4°C; the optimal antibody dilution ratio should be pre-determined ?by experimentation. Set up negative controls by omitting the primary antibody ?incubation step for one slide per each experimental condition.
Following primary antibody incubation rinse slides three times with 1x?TBST ?for ?3 minutes each.
Proteintech routinely uses EnVision Kit reagents (Dako) for this step.
Apply sufficient peroxidase labeled polymer and incubate for 30 minutes.
Rinse slides three times with 1x?TBST for 3 minutes each.
Prepare an appropriate volume of substrate solution prior to use by mixing ?one drop of Liquid DAB plus chromogen immediately with 1 ml of substrate ?buffer.
Apply the substrate carefully and incubate for 5–10 minutes till a brown ?color develops.
Rinse sections gently with sufficient distilled water.
To stain nuclei, immerse slides in a bath of hematoxylin for 3 minutes.
Rinse slides gently with distilled water.
Transfer slides into a 1% HCl, 99% ethanol solution for 10 seconds; transfer ?to distilled water immediately
Immerse slides sequentially into 60%, 80%, 95% and 100% ethanol baths ?for 5 minutes each.
Immerse slides in xylene for 5 minutes. Repeat this step again in fresh xylene ?for 5 minutes.
Mount the section with sufficient mounting media and cover with a cover slip.
Air-dry in a well-ventilated area (e.g. fume hood).
Citrate buffer | For 1000 ml | 1x TBS | For 1000 ml |
10 mM Trisodium
citrate+2H?O
|
2.9 g | 20 mM Tris-base | 2.4 g |
1.9 mM Citric
acid+H?O
|
0.4 g | 150 mM NaCl | 8.7 g |
Adjust pH to 6.0 | ? | Adjust pH to 7.6 | ? |
Add ddH?O
to 1000 ml
|
? |
Add ddH?O
to 1000 ml
|
? |
Tris-EDTA?(TE)? buffer |
?For 1000 ml | ?1x TBST | For 1000 ml |
?10 mM Tris-base | ?1.21 g | ?1x TBS | ?999 ml |
1 mM EDTA
C??H??N?Na?O??
+ 2H?O
|
?0.372 g | ?Tween-20 | ?1 ml |
Adjust pH to 9.0 | ? | ? | ? |
Add ddH?O?to 1000 ml
|
? | ? | ? |
For more IHC technical support contact us via LiveChat.
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Immunofluorescence (IF) staining is a widely used technique in biological research and clinical diagnostics. IF utilizes fluorescent-labeled antibodies to detect specific target antigens. Followed by imaging, it is a very direct technique as you can visualize results. Although it is a well-established tool, multiple factors have to be considered and various optimization steps have to be taken to ensure successful staining.
All steps in this protocol are performed at room temperature unless otherwise indicated. For optimum?staining, incubations should be carried out on a slow-moving rotary shaker unless the cell line being ?used is delicate (e.g. neuronal cells). ?Recipes for solutions highlighted bold are provided following the protocol.
Aspirate medium, wash cells seeded on clean glass cover slips briefly with ?1X PBS.
Fix the cells with 4% paraformaldehyde made fresh in 1X PBS ?for 10 minutes.
Rinse cover slips with 1X PBS ?3 times for 3 minutes each.
Permeabilize with 0.2% Triton X-100 made in 1X PBS for 5 minutes. Rinse cover?slips 3 times with 1X PBS for 3 minutes each.
Aspirate the blocking solution and apply primary antibody diluted in antibody dilution buffer. Set aside one cover slipper experimental condition for a negative control and incubate in antibody dilution buffer minus the primary antibody. ?Leave these incubations for 1 hour, or, alternatively, incubate overnight at 4°C.
Please Note: If incubating overnight, take measures to ensure the cover slips do?not dry out.
Wash cover slips with 1X PBS?3 times for 3 minutes each.
Apply an appropriate fluorophore-conjugated secondary antibody diluted in ?antibody dilution buffer ?to the coverslips and incubate for 1 hour in a moist, ?dark environment.
Please Note: It is imperative that cover slips be kept in dark conditions as much ?as possible after the addition of fluorescent reagents.
Wash cover slips with 1X PBS?3 times for 3 minutes each.
Mount cover slips on microscope slides with Hydromount (National Diagnostics) containing DAPI (if desired) for nuclear staining.
Examine slides under a fluorescence microscope.
1X PBS | In 1000 ml (final volume) |
10 mM Na?HPO? | 1.42 g |
1.8 mM NaH?PO? | 0.22 g |
140 mM NaCl | 8.18g |
2.68mM KCL | 0.20 g |
Adjust to pH 7.4 | ? |
Add ddH?O to 1000 ml | ? |
Antibody dilution buffer | In 20 ml (final volume) |
1% BSA | 0.2g |
Add 1X PBS to 20ml | ? |
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For more IF technical support download the step-by-step protocol guides below or contact us via LiveChat.
Immunoprecipitation (IP) is an affinity purification technique. IP purifies an antigen using a specific antibody that is immobilized to a solid matrix. IP is one of the most commonly used methods for isolation of proteins out of cell or tissue lysates.?
Keep samples as cool as possible by carrying out the steps below on ice or in a 4°C cold room. ?Recipes for all solutions highlighted ?bold ?are included at the end of the protocol.
Tip 1:? High concentrations of detergents interfere with immunoprecipitation (IP). Lyse cells with as small a volume volume of RIPA lysis buffer as possible before diluting the lysates with 1x PBS to the desired final volume.
Tip 2:? Use sufficient lysate: for each IP aim to use between 1–3 mg total protein. Lysates of 0.2–0.5 ml, containing a total of 1–3 mg protein, are ideally suited to a single IP. ?Measure the total protein amount by protein assay, such as Bradford or BCA assay.
Tip 3:? Make sure protease inhibitors are present in the lysate buffer. The concentration of protease inhibitor should be 1.5–2 times that of a typical lysate preparation protocol ?for ?Western blotting.
Tip 4: ?Carefully cut the end of your pipette tip at a 45° angle using a sharp blade to facilitate pipetting the bead slurry. To maintain suction, only a very small section of pipette tip needs to be removed.
Tip 5: ?Pre-clearing with Protein A or G sepharose beads is recommended for tissues ?abundant in IgG.
Add an appropriate amount (1–4 μg) of primary antibody to the whole ?(or pre-cleared) lysate. Optimal antibody concentration should be determined ?by titration. Set up a negative control experiment with control IgG (corresponding ?to the primary antibody source). Gently rock the incubations at 4°C for 2–4h ?or overnight.
Add 50 μl Protein A or G sepharose bead slurry to capture the immunocomplex. ?Gently rock the mixture at 4°C for 1–4 h.
Centrifuge the IP mixture at 1000rpm for 30 seconds at 4°C and discard ?the supernatant.
Wash the beads 3–4 times with 1 ml 1x TBST with 1x Protease inhibitor, ?centrifuge and discard the supernatant as in step 6. Keep about 80μl ?supernatant after the last centrifuge.
Resupend the pellet with 20μl ?5x SDS Sample Buffer, gently vortex for ?several seconds. Heat at 95–100oC for 5 min and centrifuge at 10,000g X g ?(approximately 9700 rpm for rotors of a 9.5 cm radius) for 3 minutes.
Load supernatants onto an SDS-PAGE gel, alternatively, transfer the supernatant ?carefully to a fresh, well-labeled microfuge tube and store at -80°C for later use. ?(For method 2, directly store at -80°C for later use).
Separate IPs by SDS-PAGE and transfer proteins to PVDF membrane. Probe with ?appropriate antibodies.
Tip 6:? For detection of immunoprecipitated proteins by Western blotting, without ?or reduced detection of non-specific artifacts (such as the heavy and light chains of ?the immunoprecipitating antibody), detect primary antibodies using HRP-conjugated ?anti-rabbit light chain-specific(L) antibody and HRP-conjugated Protein A instead of ?traditional HRP-conjugated secondary antibodies. (Protein A has higher affinity to ?intact antibodies compared with the denatured antibodies).
RIPA lysis buffer | For 1000 ml |
50 mM TrisHCl, pH 7.4 (1 M stock) | 50 ml |
150 mM NaCl | 8.76 g |
1% Triton X-100 | 10 ml |
0.5% Sodium Deoxylcholate | 5 g |
0.1 % SDS | 1 g |
10 mM NaF | 0.41 g |
1 mM EDTA (0.5 M stock) | 2 ml |
Add ddH?O to 1000 ml | ? |
Adjust to pH 7.4 | ? |
Add PMSF to 1 mM and other protease inhibitors immediately prior to use.? |
5X SDS sample buffer | ? |
250 mM Tris HCl (pH 7.0) (1M stock) | 12.5 ml |
35% Glycerol | 17.5 m |
10% SDS | 5 g |
0.02% Bromophenol Blue | 10 mg |
10% ?-mercaptoethanol | 5.0 ml |
Add ddH?O to 50ml, aliquot and store at -20°C |
For more IP technical support, contact us via LiveChat.
Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation used to investigate regions of the genome associated with a target DNA-binding protein, or conversely to identify specific proteins associated with a particular region of the genome. It is commonly used in epigenetics research (e.g., ChIP monitors transcriptional regulation via modifications of histones) .
1.?Block the reaction with 500 μl Glycine 2.5 M (final concentration 0.125 M). Incubate for 5 minutes at room temperature.
2.?Transfer the cells to a 50 ml falcon and centrifuge?at 2500 rpm for 5 minutes at 4oC.
3.?Discard the supernatant and wash twice with ice-cold?PBS ph 7.4 and centrifuge at 2500 rpm for 5 minutes?at 4oC after each washing.
4.?Resuspend cells in 5 ml Cell Lysis Buffer supplemented?with protease inhibitor. To facilitate the cell membrane?breaking, pass the lysate 3 times to a douncer. Incubate?for 15 minutes at 4oC.
5.?Centrifuge at 4000 rpm for 5 minutes at 4oC.?Discard the supernatant.
6.?Resuspend nuclei in Cell Lysis Buffer.
7.?Divide the sample into small aliquots and sonicate?for 15 minutes (high power; 30 seconds sonication,?30 seconds rest).
8.?Centrifuge at 12000 rpm for 10 minutes at room?temperature to remove nuclear debris. Discard the?pellet. Repeat this passage until the pellet cannot?be detected. Store the samples at -20oC.
1.?De-crosslink chromatin by incubating samples at?65oC for 4 hours (results may improve with an?overnight incubation).
2.?Incubate for 30 minutes with Proteinase K 50 μg/ml?final concentration at 42oC.
3.?Add 1 volume of Phenol:Chloroform:Isoamyl Alcohol?(25:24:1). Mix with vortex and let samples stand at?room temperature for 2–3 minutes.
4.?Centrifuge at 12000 rpm for 5 minutes and transfer?the aqueous phase to a new tube.
5.?Add 1/5 volume of AcNH4 10 M and 2.5 volumes of EtOH?100%. Mix and let the DNA precipitate for at least 30?minutes at -20oC.
6.?Centrifuge at max speed for 15 minutes at 4oC.?Discard the supernatant.
7.?Wash with 70% ethanol and centrifuge at max speed?for 15 minutes at 4oC. (Try to discard as much supernatant?as you can without touching the pellet.)
8.?Resuspend with TE buffer and pipette until?complete dissolution.
9.?Incubate for 30 minutes at 37oC with RNAse A at a final?concentration of 50 μg/μl.
10.?Prepare a 1.5% agarose gel.
1.?Take 70 μl of Magnetic Beads for each sample to be?immunoprecipitated. (Take the extra volume in excess:?0.5–1 times more.)
2.?After precipitation with a magnet, discard the supernatant?and wash twice with 600 μl at 5% BSA/PBS.
3.?After the second wash reconstitute the initial volume?(70*No of samples μl). (Take the extra volume in excess:?0.5–1 times more.)
4.?Take 20 μg (dependant on the tissue/cell type) for each?sample, dilute the chromatin 1:10, and bring to a final?volume of 1 ml with dilution buffer.
5.?Take 25 μl of beads for each sample and add them to?the chromatin for the pre-clearing step.
6.?Divide the remaining beads into 45 μl aliquots. Add the?corresponding antibody to each tube, plus a negative?control (specific IgG). Incubate overnight at 4oC in a?rotating wheel.
7.?Discard the beads from the chromatin samples by?putting the tubes in the magnet.
8.?Wash the Ab-Bead complexes twice with ice-cold 300 μl?at 5% PBS/BSA. Spin after the second wash and remove?the supernatant.
9.?Add 1 ml of the chromatin to each sample and resuspend?with the tip. Incubate for 2 hours at 4oC in a rotating wheel.
10.?Spin the samples and put them on the magnet.
11.?Wash twice with 1 ml low salt buffer.
12.?Wash twice with 1 ml high salt buffer.
13.?Wash twice with 1 ml LiCl buffer.
14.?Wash twice with 1 ml TE when adding the second?washes. Change tubes for new ones.
15.?Remove last wash almost completely with the pipette.
16.?Prepare Elution Buffer (EB) and set the thermomixer?to 65oC.
17.?Add 100 μl of Elution Buffer to each sample. Incubate?for 10 minutes at 65oC in the thermomixer.
18.?Put the supernatant in a new tube and repeat step 11?to obtain a final volume of 200 μl.
19.?Take 50 μl of the exceeded chromatin from the?pre-clearing as a 5% input. Add 150 μl Elution Buffer?to reach a 200 μl final volume.
20.?Incubate samples and inputs at 65oC overnight?to de-crosslink.
1.?Add 1 μl of Proteinase K to reach 50 μg/ml final?concentration. Incubate at 42oC for 1 hour.
2.?Purificate DNA by?Purification Kit .
2.?Analyze?DNA information by PCR.
Western Blotting is an analytical Immunoblotting Technique to detect specific proteins in a cell extract or tissue homogenate. Western Blotting relies on the specific binding between the protein-of-interest and an antibody raised against this particular protein.
All steps are carried out at room temperature unless otherwise indicated. Recipes for all solutions highlighted bold are included at the end of the protocol.
Tip 1: Tris-tricine gels separate low MW proteins (<20 kDa) better than Tris-glycine gels.
Blog: Tech Tips | In search of low molecular weight proteins |
Prepare samples in microfuge tubes. Add ??4X SDS sample buffer? so the total ?protein amount is 30–50 μg per sample (according to the protein amount ?measured by Bradford or BCA protein assay).
Flick microfuge tubes to mix samples, and then heat to 95-100°C for 5 minutes.
Set up electrophoresis apparatus and immerse in ?1x running buffer. Remove ?gel combs and cleanse wells of any residual stacking gel.
Load samples and protein markers onto the gel using gel loading tips. Set electrophoresis power pack to 80V (through the stacking gel), before ?increasing it to 120V when the protein front reaches the separation gel.
Tip 3: Load generous volumes of sample for the first experiment and adjust as necessary ?after assessing the initial target signal.PVDF membranes (or PSQ membranes with 0.22 μm micropores when MW ?of target is <30 kDa) are strongly recommended. Soak membranes in methanol ?for 30 seconds before moving to ?transfer buffer. Soak the filter papers and ?sponges in ?transfer buffer? as well.
Sequentially assemble the transfer constituents according to the illustration? on page 7 of this booklet and ?ensure no bubbles lie between any of the layers. Apply semi-dry ?or wet transfer systems according to the manufacturer’s instructions.
Tip 4: If target MW is larger than 100 kDa, wet transfer at 4°C overnight is suggested ?in place of a semi-dry method; moreover, we recommend adding 0.1% SDS to the wet transfer buffer to facilitate transfer.
After transfer, wash the membrane twice with distilled water, and using a pencil, mark bands of the MW ladder on the membrane. If desired, stain the membrane with commercial Ponceau red solution for 1 min to visualize protein bands, then ?wash any Ponceau red staining with copious amounts of ?1x TBST.
Block with 1x TBST containing (2-5%) nonfat dry milk (or 1-5% BSA for the ?detection of phospho-epitope antibodies) with constant rocking for 1 hour or overnight at 4°C.
Dilute primary antibody in blocking solution with a starting dilution ?ratio of 1:1000. (Optimal dilutions should be determined experimentally.) Incubate the membrane with primary antibody for 1 hour at room temperature, ?or overnight at 4°C.
Wash membrane three times with 1x TBST for 10 minutes each.
Incubate the membrane with a suitable HRP-conjugated secondary antibody (recognizing the host species of the primary antibody), diluted at 1:5000–1:50000 in blocking solution. Incubate for 1 hour with constant rocking.
Wash membrane three times with 1x TBST for 10 minutes each.
Tip 6: Do not let the membrane dry at any stage of the blotting process.
Tip 7: For preservation of the primary antibody solution over long incubations, 0.02%?NaN? could be included in the antibody dilution buffer. Not suitable for use ?with secondary antibody solutions.
Prepare ECL substrate according to the manufacturer’s instructions.
Incubate the membrane completely with substrate for 1–5 minutes (adjust time for more sensitive ECL substrates e.g. SuperSignal West Femto Chemiluminescent Substrate [Pierce]).
Expose the membrane to autoradiography film in a dark room or read using a ?chemiluminescence imaging system.
Tip 8: Use multiple exposure lengths to determine the optimal exposure time. ?Use fluorescent markers as a guide for blot-film orientation.
Line up the developed film in the correct orientation to the blot and mark the ?bands of the MW ladder directly onto the film. It is also advised to add notes ?such as lane content, film exposure time and ECL properties.?
4X SDS sample buffer | ? |
150 mM Tris?HCl (pH 7.0) (1M stock) | 15 ml? |
25% Glycerol | 25 ml |
12% SDS | 12 g |
0.05% Bromophenol Blue | 0.05g |
6% ?β-mercaptoethanol
|
6 ml |
Add ddH?O to 100ml, aliquot and store at -20°C | ? |
1X TBST | ? |
20 mM Tris-base | ?2.42 g |
150 mM NaCl | ?8.76 g |
50 mM KCl | ?3.73 g |
0.2% Tween-20 | ?2 ml |
Adjust pH to 7.6 | ? |
Add ddH?O to 1000ml | ? |
Wet transfer buffer | ? |
25 mM Tris-base | 3.03 g |
192 mM Glycine | 14.4 g |
20% Methanol | 200 ml |
Add ddH?O to 1000ml | ? |
Semi-dry transfer buffer | ? |
48 mM Tris-base | 5.81 g |
39 mM Glycine | 2.93 g |
0.0375% SDS | 0.375 g |
20% Methanol | 200 ml |
Add ddH?O to 1000ml | ? |
For target proteins with MWs between 20 and 200 kDa, make a conventional SDS-PAGE gel using the following recipes in the table below. Select the percentage of gel you require using the MW of your target protein.
Separating gel (ml, total 10 ml) | ? | ? | ? | ? |
MW of target ?protein (kDa)
|
80-200 | 35-100 | 25-60 | 20-40 |
Gel percentage | 8% | 10% | 12% | 15% |
ddH?O | ?2.1 | ?1.5 | ?0.8 | ?0 |
30% ?Acrylamide
|
?2.7 | ?3.3 | ?4 | ?5 |
2x Separating ?buffer
|
?5.0 | ?5.0 | ?5.0 | ?5.0 |
10% APS | ?0.1 | ?0.1 | ?0.1 | 0.1? |
TEMED | ?0.01 | ?0.01 | ?0.01 | ?0.01 |
Stacking gel (ml) | 4 ml | 6ml | 8ml |
MW of target ?protein (kDa)
|
- | - | - |
Gel percentage | 4% | 4% | 4% |
ddH?O | 1.4 | 2.1 | 2.7 |
30% Acrylamide | 0.5 | 0.8 | 1.1 |
2x Stacking buffer | 2.0 | 3.0 | 4.0 |
10% APS | 0.04 | 0.06 | 0.08 |
TEMED | 0.004 | 0.006 | 0.008 |
2x Separating Buffer Recipe (makes 1000ml) | ? |
Tris HCl (pH 8.8) | 90.8 g |
SDS | 2.0 g |
Dissolve compounds thoroughly. Adjust pH slowly to pH 8.8 with concentrated HCl, then add ddH2O to 1000ml. |
2x Stacking Buffer Recipe (makes 1000ml) | ? |
Tris HCl (pH 6.8) | 30.35 g |
SDS | 2.0 g |
Dissolve compounds thoroughly. Adjust pH slowly to pH 6.8 with concentrated HCl, ?then add ddH2O to 1000ml.
|
1x Running Buffer Recipe (makes 1000ml) | ? |
Tris-base | 1.51 g |
Glycine | 7.5 g |
SDS | 0.5 g |
Dissolve compounds thoroughly, then add ddH?O to 1000 ml. |
For target protein with MWs of less than 20 kDa, a tricine gel system will obtain ?higher resolution and is highly recommended. Make three layers of tricine gels as ?laid out in the following table and diagram. Apply specific tricine gel running buffer to the running system and perform transfer as usual.
Reagents | Stacking | Intermediate | Separating |
Gel percentage | 4% | 10% | 15% |
Gel volume | 2ml | 3ml | 6ml |
38% Glycerol | - | - | 1.6 |
ddH?O | 1.4 | 1.2 | - |
30% Acrylamide | 0.3 | 0.8 | 2.7 |
3.0 M Tris HCl?(pH 8.5)
|
- | 1 | 2.14 |
1.0 M Tris HCl?(pH 6.8)
|
0.3 | - | - |
10% SDS | 0.02 | 0.03 | 0.06 |
10% APS | 0.02 | 0.03 | 0.06 |
TEMED | 0.002 | 0.003 | 0.003 |
Antibody purification is performed using antigen-coupled sepharose, meaning only those antibodies specific to the immunogen are purified from the serum.
Dialyze 1 mg of fusion protein in coupling buffer overnight at 4°C.
Calculate the amount of cyanogen bromide (CNBr)-activated Sepharose4B needed ?for protein coupling. Usually, 1 g of CNBr activates 3.5 ml of Sepharose beads and?1 ml of activated Sepharose beads may absorb 5–10 mg of protein.
Activate the Sepharose beads in 20–50 ml cold 1 mM HCl for 15 min at 4°C.
Wash the beads with 1 mM HCl. In general, 1 g of Sepharose beads requires 200 ml ?of HCl to wash.
Incubate appropriate amounts of activated Sepharose beads with dialyzed fusion?proteins for 2 hrs at room temperature or overnight at 4°C.
Wash protein-coupled Sepharose matrix with 15ml ?of coupling buffer.
Add 5 ml 0.1 M Tris-HCl buffer (pH 8.0) or 1 M Ethanolaniba to block the ?uncoupled sites on beads and let stand for 2 h at room temperature or overnight at 4°C.
Wash the beads at least three cycles with acid and alkali buffer alternative.?(0.1 M Acetic/Sodium Acetate, 0.5 M NaCl, pH4.0; 0.1 M Tris-HCl, 0.5 NaCl, pH 8.0).
Incubate the beads with serum for 1–2 h at room temperature or overnight?at ?4°C.
Collect the flow-through from the purification column and save it for ELISA testing.
Wash the beads 3 times with 10 ml PBS buffer.
Wash the column with 10 ml 150 mM NaCl-HCl (pH 5) solution.
Elute the antibodies with 6 ml elution buffer and neutralize the solution with ?saturated phosphate buffer. Usually, 1 ml of elution buffer requires 50–100 μl ?of saturated phosphate buffer depending on the temperature.
For short-term storage, keep the antibody solution at 4°C; for long-term storage, ?keep the antibody in a 50% glycerol solution with 0.02% sodium azide at -20°C
Wash beads 3 times with 15 ml 0.01 M Tris-HCl (pH 7.5) buffer.
Wash beads 3 times with 10 ml of PBS buffer.
Add 2 ml PBS, 3 ml Glycerol with 0.02% sodium azide to the beads and store at?-20°C for future use.
Coupling Buffer | 1000 ml |
100 mM NaHCO? | ?8.40 g |
500 mM NaCl | ?29.2 g |
Add ddH?O to 1000 ml | ? |
Adjust to pH 8.3? |
PBS Buffer | 1000 ml |
10 mM Na?HPO? |
1.42 g
|
1.8 mM NaH?PO? | 0.22 g |
140 mM NaCl | 8.19 g |
Add ddH?O to 1000 ml | |
Adjust to pH 7.4 |
Elution Buffer | 1000 ml |
150 mM NaCl | 8.8 g |
Add ddH?O to 1000 ml | ? |
Use HCl to adjust to pH 2.5 | ? |
Saturated Phosphate Buffer |
Add Na?HPO? to PBS buffer until saturation |
For more technical support regarding antibody purification download the step-by-step protocol guides below or contact us via LiveChat.
Flow Cytometry is a technology that measures and analyzes several physical characteristics of single cells. The cells flow in a fluid stream through a beam of laser light. Using Flow Cytometry the following cell characteristics can be determined: cell size, cell granularity, complexity and relative fluorescence intensity.
Suspend cells in 1x PBS buffer and wash them twice with 1x PBS buffer by centrifugation at 1000 rpm for 5 min each time. Discard the supernatant.
Re-suspend the cells in 1 ml of 1x PBS buffer briefly.
Fix the cells in a final concentration of 4% formaldehyde (or paraformaldehyde) ?for 20 min at room temperature.
Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5 min ?each time.
Permeabilize cells by adding 100% cold methanol slowly to pre-chilled ?cells to a final concentration of 90% methanol before incubating for 30 min?on ice.
Alternatively, fix the cells in a final concentration of 4% formaldehyde ?(or paraformaldehyde) for 20 min at room temperature. Then incubate the cells ?in 0.1% Triton X-100 in 1x PBS buffer for 15 min at room temperature.
Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5?min each time.
Blocking: Incubate the cells with 3 ml blocking buffer for 1h at room temperature.
Add primary antibody at an appropriate dilution and incubate for 1h at ?room temperature.
Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5 ?min each time.
Add diluted secondary antibody (enzyme or fluorescein conjugated or other types) ?to the cells and incubate for 1h at room temperature.
Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5 ?min each time.
Re-suspend the cells in 0.5 ml 1x PBS buffer and analyze the results on a flow?cytometer. For DNA staining, re-suspend the cells in 0.5 ml of DNA dye instead; ?incubate for at least 5 min at room temperature before analyzing the results on ?a flow cytometer.
Blocking Buffer | 1000 ml |
Bovine serum albumin | 5.00 g |
1x PBS buffer | 1000 ml |
PBS Buffer | 1000 ml |
10 mM Na?HPO? | 1.42 g |
1.7 mM NaH?PO? | 0.20 g |
140 mM NaCl | 8.19 g |
Add ddH?O to 1000 ml | |
Adjust to pH 7.4 |
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Cell lysis is the breaking down of the cell membrane and the separation of proteins from the non-soluble parts of the cell. Lysate buffers contain different detergents that help to release soluble proteins (Triton-X, Tween, SDS, CHAPS). Dependent on the location of the protein of interest, a different lysate buffer is needed to obtain a high yield and purity of the protein.
Recipes for all solutions highlighted ?bold ?are included at the end of the protocol.
Pre-cool a refrigerated centrifuge to 4°C. Pellet the cultured cells by centrifugation for 5 minutes at 1000 x g (approximately 2000 rpm) at 4°C. Wash 3 times with ice-cold 1X PBS ?and then add chilled RIPA buffer? with ?protease inhibitor. In general, add 100μl RIPA buffer for approximately every 106? ?cells present in the pellet (count cells before centrifugation). Reduce the ?volume of ?RIPA buffer ?accordingly if a higher protein concentration is required. Vortex to mix and keep on ice for 30 min, vortexing occasionally.
Dissect the tissue of interest and wash briefly with chilled 1X PBS ?to remove ?any blood if necessary, cut the tissue into smaller pieces whilst keeping it on ice. Transfer the tissue to a homogenizer and add RIPA buffer with protease inhibitor. In general, add 500μl RIPA buffer for approximately every 10 mg ?of tissue. Homogenize thoroughly and keep the sample on ice for 30 min. Vortex occasionally.
Tip 1: ??Add phosphatase inhibitors to lysis buffers for extraction of phosphorylated proteins.
Sonicate the sample to break the cells or tissue up further and to shear DNA. Adjust sonication time to your type of sample: 1 min for cell lysates ?and 2–5 min for tissue lysates at a power of about 180 watts (in rounds of 10 seconds sonication/10 seconds rest for each cycle). Keep the sample on ice ?during the sonication.
Tip 2:? ?The addition of DNase for DNA digestion is not recommended as this introduces protein contamination from the enzyme.
Centrifuge at 10,000 x g (approximately 9700 rpm for rotors of a 9.5 cm radius) ?for 20 minutes at 4°C to pellet cell debris, and then transfer the supernatant to ?a fresh microfuge tube without disturbing the pellet.
Determine protein concentration of the lysate by Bradford or BCA protein assay.
Samples can be frozen at -80°C for long-term storage, or be used for immediate Western blotting or immunoprecipitation.
For Western blotting, mix sample with 4X SDS sample buffer to a final dilution?of 1X. Heat the mixture to 95°C for 5 minutes before loading onto an SDS-PAGE gel.
1X PBS | For 1000 ml |
10 mM Na?HPO? | 1.42 g |
1.8 mM NaH?PO? | 0.22 g |
140 mM NaCl | 8.19 g |
Adjust pH to 7.4 | ? |
Add ddH?O to 1000 ml |
RIPA buffer | For 1000 ml |
50 mM Tris?HCl, pH 7.4 | 50 ml |
150 mM NaCl | 8.76 g |
1% Triton X-100 or NP-40 | 10 ml |
0.5% Sodium deoxylcholate | 5 g |
0.1 % SDS | 1 g |
1 mM EDTA (0.5 M stock) | 2 ml |
10 mM NaF | 0.42 g |
Add ddH?O to 1000 ml | ? |
Add PMSF to a final concentration of 1 mM and any other protease inhibitors
immediately before use.
|
4X SDS sample buffer | For 1000 ml |
12% SDS | 120 g |
25% Glycerol | 250 ml |
150 mM Tris?HCl (pH 7.0?1M stock) | 150 ml |
0.03% Bromophenol Blue | 300 mg |
20% ?β-mercaptoethanol
|
200 ml |
Add ddH?O to 50 ml, aliquot and store at -20°C | ? |
20% ?β?-mercaptoethanol, (or 500 mM DTT replaced), should be added freshly ?before use.
? |
Inclusion bodies are aggregates of proteins. They can be found in the cell cytoplasm or nucleus. Inclusion bodies can be recovered from the cell lysates by centrifugation, extraction, and washing.?
Recipes for all solutions highlighted bold are included at the end of the protocol.
a. Suspend the cell pellet (from 1 L culture) in 30–35 ml of PBST buffer.
b. Sonicate cells in an ice-bath at 200 W for 6 min.
c. Centrifuge cell lysate for approximately 13 min at 8000 rpm, 4°C. Discard the supernatant.
d. Re-suspend the pellets in 5 ml TNMFX-2M Urea buffer before transferring to a 10 ml centrifuge tube.
e. Sonicate the solution in an ice-bath at 200 W for 1 min.
f. Add an additional 5 ml TNMFX-2M Urea to the tube. Rotate for 30 min at 4°C.
g. Centrifuge for 20 min at 4000 rpm, 4°C. Discard the supernatant.
h. Repeat steps d-g.
i. Re-suspend the pellets in 5 ml of TNMFX-0.1% Triton-X100.
j. Sonicate the solution in an ice-bath at 200 W for 1 min.
k. Add an additional 5 ml TNMFX-0.1% Triton-X100 to the tube. Rotate for 30 min at 4°C.
l. Centrifuge for 20 min at 4000 rpm, 4°C. Discard the supernatant.
m. Repeat steps i-l.
n. Vortex and wash the pellets with 2x volumes of dH?O. Centrifuge at 1000 rpm for 2 min.
o. Repeat washing until the supernatant becomes clear. Collect the pellets.
p. Dissolve the proteins depending on intended application:
- For immunization, dissolve in 1.5x volumes of 8 M urea (pH 8).
- ?For antibody purification, incubate in 2x volumes of PBS with 2% Sarkosyl overnight at 4°C. Collect the supernatant by centrifugation at 1000 rpm for 7 min.
TNMFX-2M Urea | For 1000 ml |
50 mM Tris-base | 6.06 g |
150 mM NaCl | 8.77 g |
1 mM EDTA | 0.37 g |
2 M Urea | 120.20 g |
Adjust to pH 8.0 | |
Add ddH?O to 1000 ml |
PBST buffer |
For 1000 ml
|
58 mM Na?HPO? | 8.24 g |
17 mM NaH?PO? | 2.04 g |
68 mM NaCl | 3.98 g |
1%Triton-X100 | 10 ml |
Adjust to pH 7.4 | |
Add ddH?O to 1000 ml |
TNMFX-0.1% Triton X100 | For 1000 ml |
50 mM Tris | 6.06 g |
150 mM NaCl | 8.8 g |
1 mM EDTA | 0.4 g |
0.1% Triton-X100 | 1 ml |
Adjust to pH 8.0 | |
Add ddH?O to 1000 ml |
PBS with 2% Sarkosyl | For 200 ml |
58 mM Na?HPO? | 1.65 g |
17 mM Na?HPO? | 0.41 g |
68 mM NaCl | 0.80 g |
2% Sarkosyl | 4 .00 g |
Adjust to pH 8.0 | |
Add ddH?O to 200 ml |
8 M Urea | For 200 ml |
Urea | 96.08 g |
Adjust to pH 8.0 | |
Add ddH?O to 200 ml |