Results
Fixation Prior to Immunostaining
To optimise the fixation using organic solvent and heating, we varied the temperature and used different membranes (Fig. 1). Firstly, different fixation treatments on PVDF and nitrocellulose membranes were tested (Fig. 1a). Pooled human serum sample, containing 10 μg of protein, was resolved using 10% SDS-PAGE. Following this, the samples were used for Coomassie Brilliant Blue (CBB) staining (lane i) or electroblotted onto PVDF (Fig. 1a, top panel) or nitrocellulose membrane (Fig. 1a, bottom panel), then incubated with IgG antibody after the application of different fixative treatments, including no fixation (lane ii); drying at room temperature (lane iii); heating at 50 °C (lane iv); heating at 100 °C (lane v); immersion into the organic solvents (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) (lane vi) at room temperature; immersion into the organic solvents at 0 °C (lane vii); immersion into the organic solvents at 0 °C followed by sample heating at 100 °C (lane viii) and immersion into the organic solvents at 0 °C followed by sample heating at 50 °C (lane ix). The results obtained using the PVDF membranes showed that every treatment has an effect on preventing protein loss from electroblotted membranes and increase its retaining compared to the traditional method, the difference in improving detection sensitivity between various fixation treatments and traditional method were analysed and showed in the right graph. These treatments lead to a higher intensity of the IgG signal, especially for that of the IgG light chain, a relatively low molecular weight protein. Furthermore, heating at 50 °C for 30 min (lane iv) was shown to improve IgG detection more than drying at room temperature (20–25 °C) (lane iii) or heating at 100 °C for 30 min (lane v). Acetone treatment at 0 °C (lane vii) led to better results than acetone treatment at room temperature for 30 min (lane vi), while the optimal condition of all was acetone treatment at 0 °C followed by heating at 50 °C (lane ix), both for 30 min. Furthermore, a large amount of protein was preserved on the membrane after fixation and we were able to detect degradation products of human IgG subclasses17 (asterisk, Fig. 1a, lane ix). These fragments were confirmed by analysing at least 30 μg of serum protein without the fixation step. To obtain a similar signal intensity to that of samples that underwent fixative treatment, 50 μg of serum protein was required for the analysis without applying the fixation step (Supplementary Fig. 1). We performed similar experiments using nitrocellulose membranes (Fig. 1a, bottom panel). Due to the different characteristics of nitrocellulose and PVDF membranes, different fixation procedures are required. Since nitrocellulose membranes dissolve in methanol, acetone, and other organic solvents18, we tested a series of acetone or methanol solutions, using water as the solvent. Of all the investigated fixatives, the application of 75% methanol, 50% acetone, and sample heating at 50 °C improved protein fixation the most. However, these treatments resulted in high intensity background signals. Optimal fixation was achieved when the membranes were incubated with 50% methanol/water (v/v) solution at 0 °C, followed by heating at 50 °C, both for 30 min.
Fixation method-dependent differences in the immunostaining intensity. (a) Pooled human serum samples (10 μg) were analysed by 10% SDS-PAGE, and the separated proteins were transferred onto PVDF (top) and nitrocellulose (bottom) membranes, the whole membrane was cutted into eight pieces for subsequently fixation treatments. Representative images, showing anti-human IgG antibody staining after the following treatments: lane i, Coomassie Brilliant Blue (CBB) staining; lane ii, no fixation; lane iii, drying at room temperature; lane iv, heating at 50 °C; lane v, heating at 100 °C; lane vi, immersion into the organic solvents (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) at room temperature; lane vii, immersion into the organic solvents at 0 °C; lane viii, immersion into the organic solvents at 0 °C followed by sample heating at 100 °C; lane ix, immersion into the organic solvents at 0 °C followed by sample heating at 50 °C. All treatments were performed for 30 min. The relative intensity was shown on the right (n = 3 individual experiments). (b) Proteins were separated by 10% SDS-PAGE, and transferred onto PVDF membranes, the whole membrane was cutted into five pieces for subsequently fixation treatments. Fixed in acetone at 0 °C, and heated at different temperatures for 30 min prior to the immunostaining. Lane i, CBB staining; lane ii, no fixation; lane iii, heating at 25 °C; lane iv, heating at 50 °C; lane v, heating at 75 °C; lane vi, heating at 100 °C. Right panel shows the relative intensity at different temperatures. The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. **Significantly different p < 0.01, ***p < 0.001, ****p < 0.0001. All values are means ± S.E. (error bars).
Additionally, we investigated the temperature-dependence of the PVDF fixation process (Fig. 1b), the treatments including no fixation group (lane ii); heating at 25 °C (lane iii); heating at 50 °C (lane iv); heating at 75 °C (lane v); heating at 100 °C (lane vi). Results show that retained proteins were increased in the all tested temperatures compared to the traditional method. Furthermore, results indicate that the signal intensity of IgG markedly increased at 50 °C compared to the intensity at 25 °C. However, the detection of the protein bands decreased gradually with the increase in temperature beyond 50 °C. This demonstrated that the optimal fixation temperature for detection using anti-IgG antibody is 50 °C.
Effects of the fixation step on sensitivity of immunoblotting
The sensitivity of immunoblotting coupled with the optimised fixation method was determined. Serial dilutions of the pooled human serum were separated by 10% SDS-PAGE, and the proteins were transferred onto PVDF membranes. These membranes were stained with anti-IgG, anti-haptoglobin (HP), anti-α2,6-sialyltransferases (ST6Gal1), and anti-eukaryotic elongation factor 1 alpha 2 (EEF1A2) antibodies, without pre-treatment (Fig. 2a, left) or following the optimised fixation method (Fig. 2a, right). The results showed that without the fixation step, 5 μg of serum protein was required for the visualisation of HP and IgG, two highly abundant proteins, while with the fixation step, the required amount was 1.3 μg of proteins, showing approximately four-fold increase in protein detection. The intensity of the methodology for the detection of ST6Gal1 and EFF1A2, proteins with low abundance in sera, was shown to increase two-fold following sample fixation. The intensity analysis of the retention of proteins following the fixation were shown to increase 1.8- to 16-fold, compared with those in the samples without pre-treatment (Fig. 2b). Similar results were obtained using nitrocellulose membranes (Supplementary Fig. 2), where the intensity increased approximately 1.6- to 7.6-fold.
Effects of sample fixation on the retention of proteins of the method using PVDF membranes. (a) Indicated numerals are amounts (5.0, 2.5, 1.3, and 0.6 μg) of the pooled serum proteins were subjected to 10% SDS-PAGE. The blotted membranes were treated using the traditional (left panel) or optimised fixation protocol (right). (b) Staining intensities were statistically analyzed (n = 3 individual experiments). Solid bar, no fixation; White bar, optimised fixation protocol. The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. *Significantly different p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All values are means ± S.E. (error bars).
Sample fixation prior to LB
Pooled human serum protein samples (3 μg) were used for the optimization of the fixation step coupled with the lectin blotting (LB) procedure with Lens culinaris agglutinin (LCA) and Sambucus nigra agglutinin (SNA) lectins (Fig. 3). All applied fixation methods (Fig. 3, lanes iii-vi) allowed an effective prevention of protein removal from the membranes during LB, and the difference in improving detection sensitivity between various fixation treatments and traditional method were analysed and showed in the right graph. The optimal fixation method for PVDF membranes, was acetone treatment at room temperature followed by sample heating at 100 °C, both for 30 min. In the case of nitrocellulose membranes, the optimal fixation method was a combination of incubation in 50% methanol solution and heating at 100 °C for 30 min each. More protein bands were observed while using PVDF membranes, in both cases of procedures with and without fixation, compared with those observed when using nitrocellulose membranes. This discrepancy is due to the higher mobility of low molecular-weight proteins in the nitrocellulose membranes and decreased protein binding19.
Fixation-dependent differences in lectin staining intensities when using PVDF and nitrocellulose membranes. Pooled human serum proteins (3 μg) were separated on 10% SDS-PAGE, and the proteins were transferred onto PVDF and nitrocellulose membranes, the whole membrane was cutted into five pieces for subsequently fixation treatments and followed by staining with lectins (LCA and SNA). Lane i, CBB staining; lane ii, no fixation; lane iii, drying at room temperature; lane iv, sample heating at 100 °C; lane v, organic solvent (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) treatments at room temperature; lane vi, organic solvent treatments followed by sample heating at 100 °C. All treatments were applied for 30 min. Left, WB pattern; right, quantitative analysis (n = 3 individual experiments). The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. **Significantly different p < 0.01, ***p < 0.001, ****p < 0.0001. All values are means ± S.E. (error bars).
Sensitivity of LB
The sensitivity of the LB coupled with the identified optimal fixation method was further investigated. Serial dilutions of the pooled human serum proteins were separated by 10% SDS-PAGE, and the proteins were transferred onto PVDF membranes. The membranes were stained with lectins, Phaseolus vulgaris erythroagglutinin (PHA-E), LCA, Phaseolus vulgaris leucoagglutinin (PHA-L), and Aleuria aurantia lectin (AAL), combined with either no pre-treatment or with the identified optimal fixation method (Fig. 4). All types of lectins were shown to stain more glycoproteins when the analysis was combined with the fixation step (Fig. 4a, bottom panel) than without it (Fig. 4a, top panel), which was especially prominent for the glycoproteins with small molecular weight. These glycoproteins were not detected without prior fixation. Band intensities were analysed (Fig. 4b), and the sensitivity was shown to increase from 2.8- to approximately 15-fold when the method was coupled with the fixation step. Additionally, the ability of this fixation method in retention of protein when using nitrocellulose membranes, was determined, and shown to increase approximately from 3.7- to 12-fold (Supplementary Fig. 3).
Sensitivity of the LB method coupled with the fixation step, when using PVDF membranes. Indicated numerals are amounts (3.0, 1.5, 0.7, 0.3, and 0.1 μg) of the pooled serum proteins were subjected to 10% SDS-PAGE. (a) The blotted membranes were treated using the traditional (top panel) or optimised fixation protocol (bottom panel). (b) Quantification of band intensities were statistically analyzed (n = 3 individual experiments). Solid bar, no fixation; White bar, sample fixation. The exposure times were the same for the same lectin blotting using fixation or no fixation. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. *Significantly different p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All values are means ± S.E. (error bars).
Applications of the optimised method in immunostaining and lectin staining
Immunoblotting and LB are used for the determination of protein expression and glycan level variations across different populations or conditions. CFTR, expressed in many epithelial tissues, is a key membrane protein in the complex network of molecules involved in epithelial ion transporters regulating epithelial permeability20. HIF-1, a heterodimer composed of a constitutively expressed HIF-1β subunit and a hypoxic response factor HIF-1α subunit, activates the transcription of genes that are related to critical aspects of cancer biology21. AFP is the only serum marker currently approved for clinical use in HCC diagnostics22. We not only analysed the expression levels of CFTR in HT-29 cells, HIF-1α in HEK-293T cells, GAPDH in liver tissue of mouse, and serum AFP in HCC patients, but also the glycosylation changes in the sera of prostate cancer patients, in combination with the optimised fixation method. In our analysis of CFTR expression in HT-29 cells (Fig. 5a), two bands were detected on membranes when blotting was performed either with or without the fixation step, which is consistent with a previous study23. However, for the visualisation of CFTR, the amount of total cellular protein required in no fixation group was 40 μg (Fig. 5a, top panel), while 10 μg was enough for visualisation in the optimised fixation group (Fig. 5a, bottom panel). In HIF-1α staining (Fig. 5b), the band was barely detectable even with 60 μg of protein sample in the no fixation group (Fig. 5b, top panel); in contrast, signal intensity was shown to increase eight-fold following sample fixation (Fig. 5b, bottom panel). We also tested tissue protein using this optimal fixation (Fig. 5c). The results showed that without the fixation step, 2.5 μg o tissue protein was required for the visualisation of GAPDH, while with the fixation step, the required amount was 1.3 μg of proteins, showing approximately two-fold increase in protein detection. For immunostaining of AFP (Fig. 5d), six and seven serum samples respectively obtained from healthy subjects and HCC patients were used. The increased AFP levels in the sera of the HCC patients were not detected using conventional procedure with 10 μg of total serum proteins (Fig. 5d, top panel). On the other hand, using the same amount of total protein, AFP expression was detected following the application of the fixation step (Fig. 5d, bottom panel). The elevation of serum AFP in HCC observed in our study was consistent with a previous report22.
Application of the optimised immunostaining and lectin staining methods. (a) CFTR levels in HT-29 cells. (b) HIF-1α levels in HEK-293T cells. (c) GAPDH levels in liver tissue of mouse. Various amounts (quantity represented in μg) of total cellular proteins analysed using 8% SDS-PAGE and immunostained using PVDF membrane and treated with or without fixation treatments. (d) AFP levels in the sera of healthy volunteers (n = 6) and HCC patients (n = 7), with different sample volumes using the PVDF membranes, with or without the fixation. **Significantly different p < 0.01, ***p < 0.001, ****p < 0.0001. All values are means ± S.E. (error bars). (e) AAL and PHA-E staining, using 6 μg of proteins from the sera of healthy volunteers (n = 6) and prostate cancer patients (PC, n = 10), blotted on PVDF membranes, with or without fixation. Three representative healthy samples (lane i, ii, iii) and seven representative prostate cancer samples (lane iv-x) are presented (left). Boxplot provides the quantification of the total band intensities (right). Circle, healthy subjects; square, prostate cancer patients. Student’s t-test. **P < 0.01 and ****P < 0.0001, healthy subjects vs. PC patients; #P < 0.0001, No fixation vs fixation groups. Band intensities were compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6.
Additionally, AAL and PHA-E lectins were used to analyse altered glycosylation in prostate cancer (PC) patients (Fig. 5e). The levels of fucosylated proteins and glycoproteins with bisecting glycosylation were significantly increased in the sera of prostate cancer patients, in comparison with those in the healthy subjects (P < 0.01), in both no fixation and fixation groups. The results obtained using the AAL blotting are consistent with those previously described for prostate cancer patients24. Furthermore, in both the AAL and PHA-E staining, the application of fixation led to detection of a larger number of bands than that obtained without fixation, allowing a more precise analysis of the differences between healthy and prostate cancer patient samples. The fucosylated protein (43 kDa) (framed in blue box in AAL staining) was detected in the sera of prostate cancer patients only following application of the fixation step combined with staining for AAL. In the PHA-E staining, a 40-kDa protein (framed in blue box) was detected only in the sera of the prostate cancer patients when no fixation procedure was applied, while it was detected in the sera of both healthy subjects and prostate cancer patients following the application of the optimised fixation step.
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