Multi-Peptide Strip Immunoblotting Protocol

Introduction of Multi-Peptide Strip Immunoblotting Protocol

MAPs (multiple antigenic peptides) consist of a hepta lysine backbone with end groups for anchoring multiple copies of the same synthetic peptide. Ever since MAPs were first introduced in 1988 they have been used by several investigators for eliciting antipeptide antibodies, antibodies to ribonucleoprotein complexes and in enzyme linked immunosorbent assay. The potential of MAPs as vaccines has also been investigated. In addition to these studies, MAPs have also been used in surface plasmon resonance studies, double immunodiffusion, affinity column purification, and calcium binding assays.

Multiple antigenic peptides (MAPs) can be efficiently separated on sodium dodecyl sulfate (SDS)polyacrylamide gels and transferred to a nitrocellulose membrane for subsequent use in western blot. MAPs lend themselves to Coomassie and silver on SDS-PAGE as well as by Fast Green on an immunoblot. Affinity immunoblotting for analysis of antibody clonotype distribution has been carried out using MAPs.

Figure 1. MAPs from the human Ro 60 sequence analyzed on a 15 % SDS-PAGE and stained with Coomassie blue.

Other investigators have used MAPs previously in SDS-PAGE. MAPs appear to migrate as smears in these studies. However, the MAPs used in study migrated like low molecular weight proteins, as distinct bands. MAPs can be used in SDS- PAGE and immunoblot without any further modification.

Materials of Multi-Peptide Strip Immunoblotting Protocol

Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ-cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials. The experiment do not use sodium azide in reagents.

Ro 60 multiple antigenic peptides (MAPs): Twenty one Ro 60MAPs were synthesized from the sequence of the Ro 60 autoantigen by a manual stepwise solid phase procedure. An unrelated MAP with the sequence PPPGRRPP from the Sm autoantigen was also synthesized.

15 % SDS polyacrylamide gels.

SDS PAGE running buffer: 0.025 M Tris, pH 8.3, 0.192 M glycine, 0.1 % SDS.

SDS PAGE lysis buffer: 0.3 M Tris–HCl (pH 6.8), 10 % SDS,25 % β-mercaptoethanol, 0.1 % bromophenol blue, 45 % glycerol. Leave one aliquot at 4 °C for current use and store remaining aliquots at −20 °C.

BenchMark pre-stained high and low molecular weight standards.

0.1% bromophenol blue.

Nitrocellulose membranes.

Western blot transfer buffer: 0.025 M Tris, 0.192 M glycine, 20 % methanol.

Western blot transfer apparatus: Mini Trans-Blot apparatus.

Tris buffered saline (TBS)—10× solution: 1.5 M NaCl, 0.1 M Tris–HCl, pH 7.4.

Blocking solution: 5 % milk in TBS. Store at 4 °C.

Diluent solution: 5 % milk in TBST. Store at 4 °C.

Membrane strip mini-incubation containers.

Alkaline phosphatase (AP) buffer: 100 mM Tris–HCl, pH 9.5,100 mM NaCl, 5 mM MgCl2.

Nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, p -toluidine salt (NBT/BCIP) substrate stock: Dissolve1 g NBT in 20 mL of 70 % dimethylformamide (DMF). Dissolve 1 g BCIP in 20 mL of 100 % DMF. Store at 4 °C.

Working substrate solution: Add 66μL of NBT to 5 mL of AP buffer and then add 33μL BCIP. Make up to 10 mL with AP buffer. Prepare this just prior to adding to membrane.

0.1% Fast green in 25 % methanol and 10 % acetic acid.

Methods of Multi-Peptide Strip Immunoblotting Protocol

Immunoblotting

Perform regular 15 % SDS PAGE (preparative gel or 10-well gels) and transfer to nitrocellulose membrane by Towbin's electrophoretic transfer method.

Stain with fast green to visualize the MAPs transferred to the membrane.

Trim the edges of the membrane. Wrap the fast green stained membrane in cling wrap and make a photocopy for record keeping purposes.

Excise the lane (for strip immunoblotting) with molecular weight standards from the main nitrocellulose sheet containing the transferred protein. Excise a tiny wedge from the bottom left side of the marker lane and the main membrane sheet for orientation purposes.

Excise thin strips, about 2 mm in width, from the main nitrocellulose membrane sheet. Cut a tiny wedge from the bottom left side of the strips for orientation purposes. Place them in numbered 12-lane plastic trays with the antigen side up.

Destain strips with TBST. Decant TBST once it becomes colored and replace with fresh TBST if necessary.

Block for 1 h with 1 mL each of blocking solution. Decant blocking.

Add 1 mL primary human or mouse sera at 1:100 dilution and incubate for 2 h. Decant.

Rinse strips with deionized water two to three times.

Wash 5× with TBST, 5 min each time. Dilute anti-human or anti-mouse IgG conjugated to alkaline phosphatase 1:5,000 with diluent and add 1 mL to each strip. Incubate for 1 h. Wash 5× with TBST, 5 min each time. Add 0.5 mL substrate and develop bands.

Align the strips (with developed bands) on immunoblotted strips on card board insert.

Notes of Multi-Peptide Strip Immunoblotting Protocol

The peptide sequences were designed based on the regions on Ro 60 bound by anti-Ro 60 human sera, obtained from systemic lupus erythematosus patients using, in a PIN ELISA. The MAPs were quick HPLC purified and showed a single peak on mass spectrometric analysis. In experiments varying the amount of MAP, as little as 250 ng of MAP could be detected on an immunoblot.

Four to 20 % gradient gels can also be used.

SDS precipitates at 4 °C. Therefore, the lysis buffer needs to be warmed prior to use.

Add 100 mL of 10× TBS to a 1 L graduated cylinder and make it to about 800 mL with water. Transfer 50 g skim milk powder into the cylinder and mix stir until dissolved. Make to 1 L with water. Separate 500 mL as the blocking solution. To the remaining 500 mL add 250 μL of Tween 20 (cut end of blue tip to aspirate Tween 20 easily), dissolve and use it as diluent.

Dissolve NBT in the bottle it came in. Likewise for BCIP. We started using stabilized ready-to-use BCIP/NBT substrate solution prior to switching to ECL plus chemiluminescence.

Do not heat MAPs with the lysis buffer, since it makes the MAPs to migrate as doublets at about 100,000 molecular weight on SDS-PAGE. Since they contain short sequences, there is no need to denature them with heat.

Good contact between membrane and gel is essential for good transfer. Add an extra foam if contact is insufficient.

Using a dilute fast green solution help to prevent overstaining of the membrane. Dilute stock fast green solution in gel destaining solution (25 % methanol, 10 % acetic acid). Use TBST to destain the membrane if it is overstained with fast green.

Strips can be excised very nicely using a razor blade. A glass plate is placed at an angle on the nitrocellulose at a distance of 2 mm from the edge and the razor is used to cut the strip (pull the razor blade along the sides of the glass plate to cut).

The tray can be gently slanted and the entire buffer can be discarded into the sink. The membrane strips remain stuck to the plates.

Decant diluted human sera into container having the diluted germicidal detergent Vesphene. Let sit in this for a couple of hours and then discard into sink.

Rinsing the membrane strips with deionized water two to three times will help remove a bulk of the nonspecific antibodies and other contaminants. This will help reduce the amount of TBST used subsequently and also reduce the number of washes. This wash helps to reduce nonspecific binding of NBT/BCIP to the strip. The water, owing to its low ionic strength compared to TBST, will be able to remove contaminants much better than TBST. Water is much cheaper compared to TBST, in terms of money and labor. Other investigators have found no reduction in detection of specific signals due to washing with water.

Using paper boards placed in between stacks of ELISA plates in packages of ELISA plates for this purpose.

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Multi-Peptide Strip Immunoblotting Protocol

Introduction of Multi-Peptide Strip Immunoblotting Protocol

Materials of Multi-Peptide Strip Immunoblotting Protocol

Methods of Multi-Peptide Strip Immunoblotting Protocol

Immunoblotting

Notes of Multi-Peptide Strip Immunoblotting Protocol

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