1. Introduction

Slot blot methodology, which is closely related to dot blot methodology, is a technique that immobilizes samples of DNA, RNA, or protein onto a membrane in a slot (or dot) shape,¹ without prior separation of the sample by its molecular weight, for the rapid screening of nucleic acids, proteins, or macromolecular complexes.^1-5 While both slot blot and dot blots are traditional techniques,^6,7 they continue to be adapted and refined for modern applications, such as the use of slot blot for the quantification of intracellular advanced glycation end-products, ^8,9  anti-platelet antibody screening of serum collected from patients who were suspected of having platelet-specific antibodies,¹⁰ detection of R loops (DNA–RNA hybrid),⁴  and detection of proteins used to diagnose Graves’ disease.¹¹ A slot blot can be completed in a short time and requires only a single antigen-specific antibody, making it a simple method for sample analysis.

Unfortunately, when assaying serum samples by immunoblotting, a significant source of interference can stem from immunoglobulins, such as immunoglobulin G (IgG).^12-14  IgG is an abundant serum antibody with two identical antigen-binding sites.¹⁵ IgGs are predominantly produced during secondary immune responses to pathogens and can activate pathways of the complement system.^15,16 The presence of IgG is an accepted limitation when using serum samples in Western blotting, as high IgG levels can lead to large, blotchy bands or smearing.^12-14 When using slot blot detection, after prior confirmation of primary antibody specificity (i.e., the ability to recognize the purified protein of interest, Figure 1A), we found that high levels of IgG in serum samples resulted in strong, non-specific bands on membranes probed with secondary antibody alone, which were almost identical to membranes probed with both primary and secondary antibodies (Figure 1B). This interference persisted despite the use of highly cross-adsorbed primary antibodies, as previously suggested, and ¹⁷ As a consequence, serum sample analyses are often performed with more complex and costly methods, such as enzyme-linked immunosorbent assays (ELISAs),¹⁸ or slot blot assays of serum proteins are preceded by immunoprecipitation steps.¹⁹

Currently, well-established methods for analyzing serum samples using rapid, cost-effective assays such as slot or dot blots are lacking. Therefore, we aimed to establish an improved slot blot protocol that eliminates non-specific staining, thereby enabling its use for preliminary or verification analyses of serum proteins. Such verification was required in our previous studies employing multiplex ELISA.^20-22 To address this limitation, we developed a simple serum-clearing protocol in which IgGs were depleted through two sequential incubations with Protein G–agarose beads prior to slot blotting. This could serve as a useful method for purifying immunoglobulins from animal or human serum.^23-25 Protein G purification is also considered advantageous, as it binds more broadly to IgG isotypes than protein A²⁶. We found that two sequential Protein G–agarose bead clearance steps eliminated the non-specific binding observed in membranes incubated with secondary antibody alone. Quantification analysis demonstrated strong correlations between serum protein levels measured by slot blotting and those obtained by ELISA. This improved protocol addresses a methodological gap and may be valuable for researchers encountering similar issues or exploring the use of slot or dot blots for serum protein analysis.

Figure 1. First attempt to examine TWEAK receptor (R) protein via slot blot using uncleared serum samples. (A) Specificity of this primary antibody was first determined by probing a slot blot membrane loaded with increasing concentrations of purified TWEAK R protein, with duplicates of the same sample loaded in column formation. After washing, the membrane was probed with an anti-TWEAK R antibody and an appropriate secondary antibody. A clear difference in protein concentration was observed. (B) Membrane loaded with 10 μg of uncleared rat serum per well (samples from three different rats were loaded into the three wells shown), then probed with the primary and secondary antibodies (left panel), or secondary antibody only (right panel). Ponceau S-stained membranes are shown below antibody-probed membranes. The presence of a signal after secondary-only probing, a membrane that should show no signal, suggests that the secondary antibody is binding non-specifically to components other than the TWEAK R protein. Thus, it was not possible to ascertain the concentration of TWEAK R protein in these uncleared serum samples due to this non-specific binding.

2. Materials and methods

2.1. Materials

2.1.1. Serum

Serum samples were obtained from 10 young adult female Sprague–Dawley rats (Charles River Laboratories, United States of America [USA]) exposed for four weeks to either sleep disturbance (Group A, n=5) or running wheel exercise (Group B, n=5), as previously described.²⁷

2.1.2. Reagents and supplies

(i) 1× phosphate-buffered saline (PBS; pH: 7.4, prepared according to Section 2.2)

(ii) 1× tris-buffered saline (TBS; pH: 7.6 prepared according to Section 2.2)

(iii) 1× 0.1% TBS–Tween (TBST; pH: 7.6, prepared according to Section 2.2)

(iv) 2% bovine serum albumin (BSA; pH: 7.0–7.4, prepared according to Section 2.2)

(v) Ponceau S staining solution (ThermoFisher Scientific™, USA)

(vi) Protein G–agarose beads (Roche Diagnostics, Germany)

(vii) Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific™, USA)

(viii) 96-well flat-bottom enzyme immunoassay plate (Bio-Rad, USA)

(xi) Western blotting filter paper (0.83 mm thick, 8 × 13.5 cm; ThermoFisher Scientific™, USA)

(x)  Nitrocellulose blotting membranes (0.2 µm pore size; Amersham™ Protran®, Germany)

(xi) Black Western blot incubation boxes (Licor, USA)

(xii) Cytokine-induced neutrophil chemoattractant-1 (CINC1/CXCL1) purified protein (#80039-RNCE, Sino Biological, USA)

(xiii) TWEAK receptor purified protein (TWEAK R/CD266; rabbit; #MA5-32339, ThermoFisher Invitrogen, USA).

(xiv) CINC1/CXCL1 recombinant polyclonal antibody (1:1,000; goat; #AF-515, R&D Systems, Inc., USA), detected with secondary antibody IRDye 800CW donkey anti-goat (1:20,000 dilution; highly cross-absorbed; #926-32214, Licor, USA)

(xv) Neurogenic locus notch homolog protein 1 (Notch-1) recombinant polyclonal antibody (1:1,000; goat; #AF1057, R&D Systems, USA), detected with secondary antibody IRDye 680CW donkey anti-goat, (1:20,000; #926-68074, Licor, USA)

(xvi) Tissue inhibitor of metalloproteinase-1 (TIMP-1) polyclonal antibody (1:1,000 dilution; rabbit; #BS-0415R, ThermoFisher Invitrogen, USA), detected with secondary antibody IRDye 680RD goat anti-rabbit (1:20,000; #926-68071, Licor, USA)

(xvii) TWEAK R (CD266) recombinant monoclonal antibody (1:1,000 dilution; rabbit; #MA5-32339, ThermoFisher Invitrogen), detected with secondary antibody IRDye 800CW goat anti-rabbit (1:20,000 dilution; #926-32211, Licor, USA).

2.2. Recipes

• 10× PBS (1 L): Dissolve disodium hydrogen phosphate (17.8 g), potassium dihydrogen phosphate (2.4 g), sodium chloride (80 g), and potassium chloride (2.0 g) in distilled water and adjust the final volume to 1 L. To prepare 1× PBS, dilute 100 mL of the 10× stock solution with 900 mL of distilled water. Verify and adjust the pH to 7.4 as necessary.
• 10× TBS (1 L): Dissolve Tris(hydroxymethyl)aminomethane (24 g) and sodium chloride (88 g) in distilled water, and adjust the final volume to 1 L. Check and adjust pH to 7.6 as necessary. To prepare 1× TBS, dilute 100 mL of the 10× stock solution with 900 mL of distilled water.
• 1× Washing buffer (0.1% TBST): Mix 100 mL of 10× TBS with 1 mL of Tween 20, and bring the final volume to 1 L with distilled water.
• Blocking buffer (2% BSA): Dissolve BSA powder (0.8 g; MilliporeSigma, USA) in 40 mL of 0.1% TBST. Mix thoroughly and allow to stand until the BSA is completely dissolved.

2.3. Equipment

(i) Bio-Dot® SF Apparatus (BioRad, USA) with 48 slot-shaped wells (7 mm × 0.75 mm)

(ii) −20℃ freezer

(iii) 15℃ refrigerator

(iv) Platform rocking shaker 100 115V (VWR™, USA)

(v) Vortex mixer

(vi) Odyssey CLx Imager (LicorBio, USA)

2.4. Software packages

• Licor Odyssey CLx Imager and its Image Studio (version 5.2)
• ImageJ (version 1.54j)
• GraphPad Prism (version 10.6)

2.5. Slot blot procedures

2.5.1. General preparation

• Prepare reagents (Section 2.2).
• Soak Western blot incubation boxes in 10% bleach for 12–24 hours to eliminate bacteria.

2.5.2. Depletion of immunoglobulin G to improve specific antigen binding

• Thaw samples and keep them on ice.
• Thaw Protein G–agarose beads and vortex gently.
• Using a wide tip pipette, add 50 μL of beads to the desired sample with 0.5 to 1 mg of total protein.
• Place on a shaker at 4℃ for 30 minutes to one hour.
• Centrifuge at 4395 x g for five minutes at 4℃ and remove supernatant; carefully transfer to a new tube. Repeat the step, remove the supernatant, and carefully transfer the pellet to a new tube.
• Repeat steps (iii) through (v) to ensure all IgGs have been cleared.

2.5.3. Protein quantification

• Perform the BCA assay by following the provided instructions from the Pierce™ BCA Protein Assay Kit.
• Use protein concentrations to determine the volume (μL) of sample needed to achieve 10 μg of protein per well.

2.5.4. Membrane preparation

• Cut the nitrocellulose membrane to the size of the gasket support plate, ensuring that it covers all wells. Cut the filter paper if needed. Cut off one corner of both the filter paper and the membrane as a fiducial marker.
• Place the membrane (inside of its paper cover) and two filter papers in 1× TBS to soak for at least five minutes.

2.5.5. Sample preparation

• Prepare 5 μL of serum sample aliquots.
• Dilute serum 1:20 by adding 95 μL of 1× PBS to 5 μL of serum and leave on ice.
• Label the Eppendorf tubes with corresponding sample or animal numbers and add 400 μL of 1× PBS to each tube. Each slot blot well holds 200 μL; thus, the 400 μL allows for replicates.
• Add the needed amount of sample to each designated tube using the BCA results. Vortex the mixture and leave it on ice.

2.5.6. Slot blot preparation

• Set up the slot blot apparatus, attaching the tube to the flow valve. Ensure that the flow valve is set to “Flow valve setting 1.” Do not turn on the vacuum or attach the sample template at this time.
• Place the two pieces of filter paper on the gasket support plate, ensuring that the cut corner lines up with the correct corner of the support plate.
• Carefully place the membrane on top of the filter papers, making sure the corners line up correctly. Refrain from touching the membrane as much as possible.
• Place the sample template on and slightly lock the sealing screws. Always tighten screws 1 and 4, followed by 2 and 3 (Figure 2).
• Turn on the vacuum and unlock the screws. Always follow the order: 1 and 4, then 2 and 3.
• Tighten once again, always following the order: 1 and 4, then 2 and 3.
• Test the suction by loading 1× TBS into the wells using a 200 μL multi-channel pipette. We suggest that an 8-channel pipette is best for this purpose. Look and listen for Adjust screws as needed. If the suction is poor, a whistling or suction sound will occur. When completely sealed, there should be very little noise. With the flow valve set to “Flow valve setting 1,” it should drain quickly.
• Each well should have received at least one 200 μL rinse/aliquot of 1× TBS. Keeping the wells wet with TBS is a good precaution to ensure even drainage.

2.5.7. Sample loading

• Turn the flow valve to “Flow valve setting 2” to cut off the vacuum.
• Apply samples to the desired wells. We suggest loading duplicate samples in column formation and different samples from the same experimental group in the same rows, as shown in Figure 2. Each well gets 200 μL of the 1× PBS/sample solution. Try to avoid touching the membrane with the pipette. Aim for the middle of each well.
• Once all samples have been applied, turn the flow valve to “Flow valve setting 3.” Manually cover the outlet for control over the suction. It should be a very gentle suction. A finger can be used to partially cover the output, but some air must still get through. This could take several minutes for all the samples to go through.
• Still using flow valve setting 3, partially cover the output with a soft lab wipe, such as a Kimwipe.
• Run 1× TBS through all wells, one last time with gentle suction.

2.5.8. Ponceau S red staining and membrane blocking

• While the 1× TBS is being suctioned through, add deionized water to the black incubation box (enough to cover the membrane), to remove remnants of TBS on the membrane.
• After all the 1× TBS has drained through, leave the vacuum on and unlock in the order of screws 1 and 4, then screws 2 and 3.
• After the sample template is off, the vacuum can be turned off.
• Use forceps to take the membrane off and place it in the deionized water in the black incubation box. Important: Always keep the membrane moist.
• Wash: Put on the rocker at room temperature for one minute at a medium-low speed. Empty and repeat for a second and third wash.
• Discard the deionized water and use a transfer pipette to put Ponceau S red stain onto the membrane. The volume required to fill one or two normal-sized transfer pipettes is usually enough to cover the membrane (2–4 mL).
• Leave on the rocker at room temperature for 5–15 minutes.
• Discard the Ponceau stain (can be saved and filtered later for reuse) and wash the membrane for one minute with deionized water several times until the water comes out clear.
• Take a photo of the Ponceau S stain. A lightbox is preferred but not required.
• Discard the deionized water and add 5–10 mL of 2% BSA onto the membrane and incubate on the rocker for 1–1.5 hours at room temperature or overnight at 4℃.

2.5.9. Antibody incubation

• Choose the desired primary antibody and dilute it (we used 1:1,000) in 2% BSA. We typically use 10 mL 2% BSA to 10 μL of primary antibody.
• Discard the blocking BSA and add the diluted antibody.
• Incubate on the rocker for 1–1.5 hours at room temperature or overnight at 4℃.
• Discard the primary antibody (can be saved and stored in a −20℃ freezer).
• Wash: Begin washing membrane in TBST 0.1%. Repeat washing three times for 10 minutes or four times for five minutes. Leave on rocker at room temperature.
• Choose a secondary antibody and dilute it as needed; we used 1:20,000. We added 1 μL of secondary antibody to 20 mL 2% BSA.
• After washing, add a secondary antibody and incubate on the rocker for 45 minutes to one hour at room temperature                                                                                                                     
• Discard the secondary antibody and wash again in 0.1% TBST three times for 10 minutes, or four times for five minutes.

2.5.10. Visualization

• Use an imager, such as the Licor Odyssey CLx Imager and its Image Studio (version 5.2) software for visualization.
• Clean the glass with deionized water and a soft lab wipe.
• Using forceps, gently place the membrane face down in the bottom left corner.
• Wet a mini roller in the TBST from the incubation box and roll out the bubbles.
• Set the analysis area on the computer screen to match the membrane area on the glass.
• Select the wavelength required to detect the secondary antibody.
• Close the lid of the imager and hit start.
• Once complete, flip the membrane face up again and place it back in the TBST in the incubation box.

2.5.11. Storage (Optional)

• Place the membrane face up in the middle of an aluminum sheet. Fold the aluminum to securely cover the membrane.
• Store in a 15℃ refrigerator in case re-imaging or re-probing is needed.
• Put the Licor boxes in a 10% bleach solution overnight to clean.

2.5.12. Analysis of slot blot band (optional)

• ImageJ (version 1.54j) was used to evaluate the pixel density of each well of the slot blot membrane.
• The whole image of each membrane was converted to grayscale before levels were balanced to highlight individual peaks.
• The area under the peaks of each well was measured using ImageJ.
  • Open the image in ImageJ (Use the drop-down box of “Open” under the “File” tab)
  • If the image is crooked, rotate to straight (“Image” tab, “Transformation,” “Rotate”)
  • With the rectangular tool in the toolbar, draw a rectangle around all bands of interest in one row. Do this for one row of bands at a time.
  • Answer the ImageJ question as to whether the rows are horizontal using either Control 1 (PC) or Command 1 (Mac).
  • Generate a histogram of the row of bands using either Control 3 (PC) or Command 3 (Mac).
  • If the image histogram is inverted, use the “Edit” drop-down tab to “Invert.”
  • Use the line tool in the toolbar to separate the peaks of the bands from the baseline.
  • With the magic wand tool in the toolbar, click on each peak to get the area under the curve.
  • Copy and paste the numbers generated by ImageJ into Excel.
• Using the steps indicated in step (iii), determine the pixel density of both the antibody-detected bands, the Ponceau S red-stained bands, and the density of blank wells (loaded with TBS only).
• Subtract the density of blank wells from the measurements of wells filled with samples.
• Normalize the density of the antibody-detected band to the density of the Ponceau S red of the same well.

Figure 2. Slot blot instrument assembly and loading. Screw numbers are labeled. Tightening and loosening must be in the same order every time: 1 and 4, followed by 2 and 3. Our preferred method is to load samples in duplicate (superior to inferior) and individual animal serum samples left to right. Serum from 10 rats (indicated by numbers in the image) was loaded with a gap between for ease of image analysis. Two groups were assayed (A and B). 

2.6. Enzyme-linked immunosorbent assay (optional)

Protein analytes in serum samples were assayed using RayBiotech’s Quantibody Rat Cytokine Array platform (QAR-CAA-67, RayBiotech, USA) as a manufacturer-provided service. All procedures were performed in-house by RayBiotech (USA) according to their protocol, antibody verification steps, and software analysis on blinded samples. Results were converted to pg/μg of total protein using BCA protein analysis.

3. Results

3.1. Retention of non-specific bands in secondary antibody-only immunostaining of centrifuged serum samples

We began our experiments by first verifying the efficacy of a specific primary antibody’s ability to recognize the protein of interest. For this initial testing, an increasing gradient of the purified protein was loaded onto the slot blot (Figures 1A & 3A) when slot blots were probed for TWEAK R or CINC1. In these examples, increasing concentrations of the purified protein resulted in stronger signals after primary and secondary antibody incubations and imaging.

      Our next antibody verification step was to run two blots with serum samples: one probed with primary and secondary antibodies, and the other with secondary antibody only, as typically recommended in immunoblotting guides.²⁸ For this subset of experiments, after collection, blood samples were centrifuged to remove cells and other debris, using previously described methods.^22,27 Serum was loaded at a concentration of 10 μg of protein per well (protein content of serum samples was determined using BCA), with different samples from the same group members loaded along the same row (left panels in Figures 1B & 3B). Ponceau S red staining of the membrane was used to confirm equal amounts of serum protein were loaded per well (red-stained bands in images below the immunoblotted membranes in Figures 1B & 3B). Disappointingly, the use of these centrifuged serum samples (i.e., without clearance of IgGs) showed that slot blots incubated with the secondary antibodies alone had nearly identical signals as slot blots incubated with primary and then secondary antibodies (right panels in Figures 1B & 3B). The presence of an immune-detected signal after probing with the secondary antibodies only (Section 2.1.2), which should show no signal,²⁹ strongly suggested that the secondary antibodies were binding non-specifically to components other than the protein of interest.^28,30 We hypothesized that this non-specific binding might be directed toward serum IgGs, given the high IgG concentration in serum.^14,31

3.2. Elimination of non-specific binding via Protein G–agarose bead-mediated immunoglobulin G removal

Therefore, we used Protein G–agarose beads to remove IgGs from the serum samples to eliminate non-specific binding (as described in Section 2.5.2). Fifty μL of Protein G–agarose beads were added to each serum sample of 0.5 to 1 mg of total protein. Serum samples with beads were incubated together on a shaker for 30 minutes at 4℃. Samples with beads were then centrifuged twice at 4395 x g for five minutes at 4℃ before removing the supernatant. Best results were achieved when the process was repeated, as described in the next section. After IgGs were depleted from the serum samples, probing the slot blot membranes with secondary antibody alone (IRDye 800CW donkey anti-goat antibody) produced no detectable signal (upper right panel, Figure 3C), in contrast to the clear bands observed when the same secondary antibody followed primary antibody targeting CINC1 (upper left panel, Figure 3C).

3.3. Achievement of optimal immunoglobulin G clearance with repeated Protein G–agarose bead incubation

Secondary antibody-only experiments were continued to determine the number of repeat incubations of serum with Protein G–agarose beads required for optimal clearance. As shown in Figure 4, three different serum samples were loaded onto the slot blot, each after no clearance, one clearance, or two clearance steps, and probed with only the IRDye 800CW goat anti-rabbit secondary antibody. Membranes loaded in duplicate with uncleared serum samples (from three different rats) showed strong signals in both color and black-and-white output images (Figure 4A & B). Serum samples that were cleared only once showed a low level of signal (Figure 4B). In contrast, serum samples cleared twice showed no signal on these secondary antibody-only-probed membranes (Figure 4A & B, the bottom row). Ponceau S red staining was used to show that equal amounts of serum protein were loaded per well (Figure 4C). Notably, a comparison of Ponceau S red staining of uncleared versus cleared samples showed a slight loss of protein after clearance.

3.4. Variations in specific protein content in slot blot membranes after immunoglobulin G clearance

In our subsequent experiments, serum samples from 10 different rats (n=5/group) were used to examine the specific content of Notch-1, TIMP-1, and TWEAK R in the individual serum samples. Uncleared samples showed nearly equivalent band staining in all membranes (Figure 5A). After IgG clearance with Protein G–agarose beads, each of the four slot blot membranes, probed with four different primary antibodies, showed variations between samples and groups, as typically expected in immunoblots with different samples and groups (Figures 3C & 5B). Figures 3C and 5C show Ponceau S red staining of the same membranes, indicating that similar amounts of protein were loaded per well. Figure 5D shows that serum samples cleared twice indicated the absence of signal in a secondary antibody-only probed membrane (probed with IRDye 680RD goat anti-rabbit).

3.5. Correlation of immunoblot band intensity with Ponceau red staining and ELISA data

We used ImageJ to determine the densities of the antibody-detected bands and Ponceau S red-stained bands on each membrane, as described in the methods section (Section 2.5.12). The density of antibody detected bands was then normalized to the density of the Ponceau S red-stained bands and multiplied by 100 (to achieve a percent density of specific detected protein versus total protein loaded in the well). These slot blot analysis results are presented in Figure 6A, after comparing group differences using unpaired t-tests with Welch’s correction. The same samples used in the slot blot assay were also used in the ELISA methodology. The ELISA results (presented as pg/μg total protein) are presented in Figure 6B (similar statistics were used to compare the ELISA data; pg of protein per mL of serum data is shown in Supplementary File 1). We then calculated Spearman’s r correlation coefficients to compare the outcomes of the slot blot with those of the ELISAs. Strong correlations (r>0.6)^32-34  were observed when comparing slot blot results to ELISA results (Figure 6C, right panels). These correlations underscore the values of this improved slot blot method. Notably, some differences were observed. These differences were likely due to the use of different specific antibodies in each method. Antibodies are known to vary due to differences in the epitopes recognized by each on the target protein, as well as monoclonal versus polyclonal differences in the detection of epitope site(s).^10,29,35  There are also differences in ELISA versus slot blot membrane methodologies that might contribute to the observed variations.^11,35

^{Figure 3. Examination of cytokine-induced neutrophil chemoattractant-1 (CINC1) via slot blot using uncleared serum samples or serum samples cleared of immunoglobulin G (IgGs) using Protein G–agarose beads. (A) Specificity of the primary antibody was first determined by probing a slot blot membrane loaded with increasing concentrations of purified CINC1 protein. After washing, the membrane was probed with an anti-CINC1 antibody and an appropriate secondary antibody. A clear difference in protein concentration was observed. (B) Membrane loaded with 10 μg of uncleared rat serum per well (sample duplicates were loaded in column formation; different samples from the same group were loaded across the same row; each group contains five different samples per group, numbered as indicated), then probed with the primary and secondary antibodies, or with the secondary antibody only. Note the non-specific secondary antibody-only staining in the right upper panel. Ponceau S-stained membranes are shown below antibody-probed membranes. (C) Slot blot membranes loaded with Protein G–agarose bead-cleared samples (10 μg per well; sample duplicates were loaded in column formation; different samples from the same group were loaded across the same row). The left panel, probed with both primary and secondary antibodies, was loaded with three different samples per group; the right panel, probed with only the secondary antibody, was loaded with five different samples per group. Ponceau S-stained membranes are shown below the antibody-probed membranes. The absence of signal on the secondary antibody-only-probed membrane (IRDye 800CW donkey anti-goat, in this case) indicates that the IgG clearance step eliminated non-specific secondary antibody-only staining.}

^{Figure 4. A repeated clearance step using Protein G–agarose beads was required to eliminate non-specific immunostaining caused by a secondary antibody (IRDye 800CW goat anti-rabbit, in this example). (A, B) Slot blot membranes loaded with uncleared serum samples (upper two rows), or samples cleared once or twice with Protein G–agarose beads (10 μg per well), as indicated. Duplicates of a sample were loaded in column formation; three different samples were loaded across the rows. Each was probed with the secondary antibody only. (C) Ponceau S-stained membranes. BW indicates black and white Licor image output.}

^{Figure 5. Examination of several other serum proteins (Neurogenic locus notch homolog protein 1 [Notch-1], tissue inhibitor of metalloproteinase-1 [TIMP-1], and TWEAK receptor [R]) via slot blot using either uncleared serum samples or samples cleared of immunoglobulin G (IgGs) twice with Protein G–agarose beads. The same 10 serum samples were loaded in duplicate, as shown, for each membrane and protein. (A) Slot blot membranes probed with both primary and secondary antibodies in uncleared serum samples, showing that each blot looked similar. Duplicates of a sample were loaded in column formation; different samples from the same group were loaded along the same row (n=5/group, numbered as indicated). (B) Slot blot membranes probed with both primary and secondary antibodies after two repeats of Protein G–agarose bead incubations. These blots now show variations between individual samples and antibody-detected proteins. Samples were loaded as indicated for Panel A. (C) Ponceau S staining of each membrane after imaging of antibody-detection steps and washing. (D) Membrane probed with only a secondary antibody, with samples loaded as indicated for Panel A. The absence of signal in the secondary antibody-only probe of the membrane (IRDye 680RD goat anti-rabbit, in this case) shows that the IgG clearance step eliminated the non-specific secondary antibody-only staining.} 

Figure 6. Comparison between slot blot and enzyme-linked immunosorbent assay (ELISA) results for each of the four serum proteins examined in this study (Cytokine-induced neutrophil chemoattractant-1 [CINC1], neurogenic locus notch homolog protein 1 [Notch-1], tissue inhibitor of metalloproteinase-1 [TIMP-1], and TWEAK receptor [R]). (A) Slot blot analyses using serum samples cleared twice of immunoglobulin G using Protein G–agarose beads. Each group contains three or five different samples per group (n=3/group for CINC1). (B) ELISA outcomes presented as picograms of antibody-detected proteins normalized to micrograms of total protein in the sample. Each group contains three or five different samples per group (n=3/group for CINC1). (C) Spearman’s r correlation with an r of more than 0.6 is defined as a strong correlation.^32-34  Statistical significance is indicated as *p<0.05, **p<0.01, and ns = not significant.

^{Abbreviation: OD: Optical density.}

4. Discussion

We developed a simple serum-clearing protocol in which IgGs were removed by incubating serum with Protein G–agarose beads twice prior to slot blotting. Twice-repeated clearance steps eliminated the non-specific binding observed with secondary antibody-only incubation of membranes. This can be easily seen in the lower right panel of Figure 3C, twice cleared bands in Figure 4A and B, and in Figure 5D, where no signal was detected in secondary antibody-only probed membranes (probed with IRDye 800CW donkey anti-goat, IRDye 800CW donkey anti-goat, and IRDye 680D goat anti-rabbit, respectively). Thus, IgG removal is critical for obtaining a specific and accurate result.

Our findings directly address a significant challenge in immunodetection: interference from endogenous antibodies, such as IgG, which are well documented as a cause of false-positive results across various immunoassay formats.^12-14,31 The lack of staining in the secondary antibody-only probed membranes, combined with image analysis and density normalization of immunodetected bands to the density of total protein loaded into each well (the latter seen after Ponceau S red staining), supports our hypothesis that slot blotting can be an effective semi-quantitative method for evaluating protein concentrations across samples. This is further supported by the strong correlations with ELISA data for the same proteins in the same samples (Figure 6). These latter results agree with similar findings demonstrating that slot blot assays and ELISA can be robust, complementary tools for cross-validation in protein–protein interaction analyses³⁶ and hepatitis C virus detection. ³⁷

However, some limitations should be considered. First, the lack of molecular weight resolution in slot blots compared to western blots limits the detection of post-translational protein modifications. Second, although efforts were made to standardize sample volumes and antibody concentrations, minor inconsistencies may have affected the reproducibility of the results. Troubleshooting is typically required for this type of assay (Table 1). In addition, there appeared to be a slight loss of total protein after the Protein G–agarose clearance steps (as indicated by the slight loss of Ponceau S red staining in cleared samples; Figure 4C). Thus, the slot blot method would be disadvantageous if the protein of interest is at a low concentration in the serum.

Despite these limitations, our findings emphasize the use of slot blotting as a fast, cost-effective alternative for screening serum samples for protein content. The technique is especially valuable in contexts where qualitative or relative quantification is sufficient, such as verifying the presence or absence of a protein in various conditions or screening clinical samples for biomarkers. This is supported by the work of Ameri et al.,¹⁰ who demonstrated that slot blots are an efficient and acceptable procedure for platelet antibody screening in a clinical cohort with high sensitivity and specificity. It may also be useful in resource-limited settings or initial biomarker screening where ELISA plate readers may not be readily available. The utility of slot blotting as a practical alternative to ELISA is further demonstrated by Japolla et al.,³⁸ who developed a nanogold-based slot blot inhibition assay for viral antibody detection that also showed strong correlation with ELISA data without the need for specialized spectrophotometry. Dot blot assays, which are closely related to slot blots, have also been deemed suitable as simple and rapid detection assays, versus ELISAs, for the detection of Graves’ disease in rural areas when ELISA detection equipment is not available (although sensitivity differences are likely).¹¹ A key step in our protocol was the effective removal of IgGs from the serum. This aligns with the emphasis in other recent slot blot methodologies on the critical importance of sample-specific optimization. For example, Takata⁸ highlighted the use of a specialized lysis buffer for intracellular Advanced glycation end-products, and Sarker and Cooper⁴ employed specific enzymatic pre-treatments (RNase H) to ensure assay specificity for nucleic acid structures.⁴

Looking forward, the reproducibility of this technique could be further enhanced by exploring different membrane types and optimizing antibody conditions.³⁹ For example, the work of Kang et al.⁴⁰ demonstrates that the basic slot blot principle can be scaled into a microarray format for high-throughput epitope screening, suggesting a promising direction for adapting this serum analysis protocol to screen multiple cytokines or patient samples in parallel.

5. Conclusion

This optimized protocol addresses a gap in the literature, guiding researchers facing similar challenges or using slot blots for serum protein analysis. These same methods could be applied to dot blot methodology. Our refinement of the slot blot method could make it a robust tool for high-throughput screening in both research and clinical settings, as well as for validating ELISA and other methods used to examine specific serum proteins.