One μL sample loading buffer (5×) (Beyotime Institute of Biotechnology, Jiangsu, China) was dissolved in 4 μL the SNC sample. The mixture was heated to 100 °C for 5 min, followed by centrifuging at 9000 g for 5 min. Subsequently, 5 μL supernatant was loaded onto an instant polyacrylamide-gel made of 10% running gel and 6% stacking gel. After electrophoresis, the gels were stained BeyoBlue™ Coomassie Blue Super Fast Staining Solution and destained with deionized water. Quantitative analysis of protein band intensity was performed using a Model GS-700 Imaging Densitometer (Bio-Red Laboratories, Hercules, CA, USA) with Molecular Analyst Software version 1.4 (image analysis systems).
Sample loading buffer 5
Manufactured by Beyotime
Sourced in China
Sample loading buffer (5×) is a concentrated solution used to prepare samples for gel electrophoresis. It helps to increase the sample density, track the progress of electrophoresis, and stabilize the pH of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Protein g agarose, by Merck Group (1 mentions)
Anti igf2bp2, by Proteintech (1 mentions)
Anti ythdf1, by Proteintech (1 mentions)
Anti igf2bp3, by Proteintech (1 mentions)
Anti ythdf2, by Abcam (1 mentions)
Anti mettl14, by BioLegend (1 mentions)
Anti ythdf3, by Abcam (1 mentions)
Bca protein quantitative kit, by Beyotime (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Anti gapdh, by Abcam (1 mentions)
3 protocols using sample loading buffer 5
1
SNC Protein Quantification Protocol
2
Immunoprecipitation of Myc- or HA-tagged proteins
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The HEK293 cells were transfected in 100-mm dishes with the indicated plasmids. Twenty-four hours after transfection, the cells were collected and washed twice with 1×PBS, resuspended gently in 450 µl IP buffer and placed on a vibrating mixer at 4°C for 20 min. The cell lysates were then centrifuged, and the supernatants were separated into two aliquots. One aliquot of 400 µl was mixed with the pre-washed protein G agarose (Merck Millipore, Darmstadt, Germany) together with 1 µg of the indicated antibodies (control mouse IgG and anti-Myc or anti-HA MAb) and placed on a rotational tumbler for incubation overnight at 4°C. The agarose was washed with IP buffer and boiled with 50 µl Laemmli sample buffer and designated as the IP sample. Another aliquot of 40 µl supernatant was boiled with sample loading buffer (5×) (Beyotime, Shanghai, China) and designated as the input sample. All of the samples were analyzed by immunoblotting.
3
Western Blot Analysis of RNA Binding Proteins
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The tissue or cell was cleaved for 20 minutes with RIPA (Beyotime) lysate on ice. After centrifugation, the protein was quantified using a BCA protein quantitative kit (Beyotime). The protein concentration was determined by the protein standard curve, and the protein was adjusted into the proper concentration. Sample loading buffer (5×) (Beyotime) was added and the mixture was boiled at 100℃ for 5 minutes. The PAGE glue was prepared according to the molecular size of the corresponding protein and electrophoretic separation was performed after adding samples. The membrane was then transferred by constant current 270 mA for 90 minutes. We blocked the membrane with 5% milk, it was incubated with primary Ab then secondary Ab, and finally scanned and recorded with an Odyssey infrared scanner, semiquantified with internal reference GAPDH. Antibodies used in this experiment were as follows: anti‐GAPDH (1:5000, Cat# ab8245, Cat#ab9485; Abcam), anti‐METTL14 (1:1000, Cat#669602; BioLegend), anti‐YTHDF2 (1:1000, Cat#ab220163; Abcam), anti‐YTHDF1 (1:1000, Cat#17479‐1‐AP; Proteintech), anti‐YTHDF3 (1:1000, Cat#ab220161; Abcam), anti‐IGF2BP2 (1:1000, Cat#11601‐1‐AP; Proteintech), and anti‐IGF2BP3 (1:1000, Cat#14642‐1‐AP; Proteintech).
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