| Western blot (WB): | 1:500-2000 |
| Immunohistochemistry (IHC): | 1:50-400 |
| Immunocytochemistry/Immunofluorescence (ICC/IF): | 1:50-400 |
| Flow Cytometry (Fixed): | 1:50-200 |
| (Boiling the paraffin sections in 10mM citrate buffer,pH6.0,or PH8.0 EDTA repair liquid for 20 mins is required for the staining of formalin/paraffin sections.) Optimal working dilutions must be determined by end user. | |

Figure 1. Western blot analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human Caco-2 whole cell lysates
Lane 2: human A549 whole cell lysates
Lane 3: human THP-1 whole cell lysates
Lane 4: human SW620 whole cell lysates
Lane 5: human U-937 whole cell lysates
Lane 6: human HepG2 whole cell lysates
Lane 7: rat RH35 whole cell lysates
Lane 8: mouse RAW246.7 whole cell lysates
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-HSPD1 antigen affinity purified monoclonal antibody (Catalog # M01280-3) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for HSPD1 at approximately 60KD. The expected band size for HSPD1 is at 60KD.

Figure 2. IHC analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
HSPD1 was detected in paraffin-embedded section of human intestinal cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSPD1 Antibody (M01280-3) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.

Figure 3. IHC analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
HSPD1 was detected in paraffin-embedded section of human lung cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSPD1 Antibody (M01280-3) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.

Figure 4. IHC analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
HSPD1 was detected in paraffin-embedded section of human mammary cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSPD1 Antibody (M01280-3) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.

Figure 5. IHC analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
HSPD1 was detected in paraffin-embedded section of mouse liver tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSPD1 Antibody (M01280-3) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.

Figure 6. IHC analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
HSPD1 was detected in paraffin-embedded section of rat liver tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSPD1 Antibody (M01280-3) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.

Figure 7. Flow Cytometry analysis of A431 cells using anti-HSPD1 antibody (M01280-3).
Overlay histogram showing A431 cells stained with M01280-3 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-HSPD1 Antibody (M01280-3, 1:100) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 1:100) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1:100) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Figure 8. Flow Cytometry analysis of HepG2 cells using anti-HSPD1 antibody (M01280-3).
Overlay histogram showing HepG2 cells stained with M01280-3 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-HSPD1 Antibody (M01280-3, 1:100) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 1:100) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1:100) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Figure 9. ICC analysis using anti- HSPD1 antibody (M01280-3). was detected in immersion fixed A431 cell line. Cells were stained using the Dylight488-conjugated Anti-mouse IgG Secondary Antibody (green)(Catalog # BA1126) and counterstained with DAPI (blue).

Figure 1. Western blot analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human Caco-2 whole cell lysates
Lane 2: human A549 whole cell lysates
Lane 3: human THP-1 whole cell lysates
Lane 4: human SW620 whole cell lysates
Lane 5: human U-937 whole cell lysates
Lane 6: human HepG2 whole cell lysates
Lane 7: rat RH35 whole cell lysates
Lane 8: mouse RAW246.7 whole cell lysates
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-HSPD1 antigen affinity purified monoclonal antibody (Catalog # M01280-3) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for HSPD1 at approximately 60KD. The expected band size for HSPD1 is at 60KD.

Figure 2. IHC analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
HSPD1 was detected in paraffin-embedded section of human intestinal cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSPD1 Antibody (M01280-3) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.

Figure 3. IHC analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
HSPD1 was detected in paraffin-embedded section of human lung cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSPD1 Antibody (M01280-3) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.

Figure 4. IHC analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
HSPD1 was detected in paraffin-embedded section of human mammary cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSPD1 Antibody (M01280-3) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.

Figure 5. IHC analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
HSPD1 was detected in paraffin-embedded section of mouse liver tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSPD1 Antibody (M01280-3) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.

Figure 6. IHC analysis of HSPD1 using anti-HSPD1 antibody (M01280-3).
HSPD1 was detected in paraffin-embedded section of rat liver tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-HSPD1 Antibody (M01280-3) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.

Figure 7. Flow Cytometry analysis of A431 cells using anti-HSPD1 antibody (M01280-3).
Overlay histogram showing A431 cells stained with M01280-3 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-HSPD1 Antibody (M01280-3, 1:100) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 1:100) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1:100) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Figure 8. Flow Cytometry analysis of HepG2 cells using anti-HSPD1 antibody (M01280-3).
Overlay histogram showing HepG2 cells stained with M01280-3 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-HSPD1 Antibody (M01280-3, 1:100) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 1:100) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1:100) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Figure 9. ICC analysis using anti- HSPD1 antibody (M01280-3). was detected in immersion fixed A431 cell line. Cells were stained using the Dylight488-conjugated Anti-mouse IgG Secondary Antibody (green)(Catalog # BA1126) and counterstained with DAPI (blue).








