Recombinant Human Pro-SUMO3 Protein, CF Summary
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins.Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration.The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard.In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
UL-760
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Reconstitution Calculator
Background: SUMO3
Human Small Ubiquitin-like Modifier 3 (SUMO3), also known as SMT3A, is synthesized as a 103 amino acid (aa), propeptide with a predicted 11.5 kDa. SUMO3 contains a two aa C-terminal prosegment. Human SUMO3 shares 83% sequence identity with mouse SUMO3. SUMO3 also has high aa sequence homology to SUMO2 and SUMO4, 87% and 75%, respectively. SUMO3 shares only 47% sequence identity with SUMO1. SUMOs are a family of small, related proteins that can be enzymatically attached to a target protein by a post-translational modification process termed SUMOylation (1-3). All SUMO proteins share a conserved Ubiquitin domain and a C-terminal diglycine cleavage/attachment site. Following prosegment cleavage, the C-terminal glycine residue of SUMO3 is enzymatically attached to a lysine residue on a target protein. In humans, SUMO3 is conjugated to a variety of molecules in the presence of the SAE1/UBA2 SUMO-activating (E1) enzyme and the UBE2I/Ubc9 SUMO-conjugating (E2) enzyme (4,5). In yeast, the SUMO-activating (E1) enzyme is Aos1/Uba2p (6). Because of the high level of sequence homology most studies report effects of SUMO2/3. For example, addition of SUMO2/3 was shown to modulate the function of ARHGAP21, a RhoGAP protein known to be involved in cell migration (7). Other reports indicate that the conjugation by SUMO2/3, but not SUMO1, may represent an important mechanism to protect neurons during episodes of cerebral ischemia (8,9). However, studies suggest that SUMO2/3 expression is regulated in an isoform-specific manner since oxidative stress downregulated the transcription of SUMO3 but not SUMO2 (10).
All SUMO isoforms are translated with additional C-terminal residues that have to be removed to generate the active protein. Pro-SUMO-3 (103 aa) is the inactive precursor of SUMO-3 (92 aa) and is processed at the C-terminus by SUMO-3 specific proteases (SENPs).The resulting SUMO-3 protein has the conserved C-terminal Gly-Gly residues that function in activation and conjugation reactions. This protein can be used as a negative control in SUMOylation reactions or as a substrate for SENPs. NCBI Accession # NM_006936.
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尤其是前面两个，后面的还可以从字面理解着做，前面这两个就纯把我搞晕菜了．
全量法：测水浸出物的
酒石酸铁比色法；
紫外分光光度法；
恒重法：测水含量的
谢谢谢谢
红色的是胰岛素，绿色的是胰高血糖素。都是石蜡切片~
请问下我这染出来的应该是胰岛细胞吧，背景颜色适合吗？
为什么胰高血糖素然不出来，这两种我用的方法是一样的。
方法：
1.脱蜡，水化：脱蜡前应将组织芯片在60摄氏恒温箱中烘烤1h
A：组织芯片置于二甲苯中浸泡15min，更换二甲苯后再浸泡10min（脱蜡）
B：无水乙醇中浸泡5min（水化）
C：95%乙醇中浸泡5min
D：75%乙醇中浸泡5min
2.PBS洗2次各5min
3.PBS洗2次每次5min，洗去枸橼酸钠（Ⅲ，Ⅱ）
4.3%H2O2(80%甲醇稀释)室温静置10min（去除内源性酶）
5.PBS洗2次各5min（Ⅰ、Ⅱ）
6.加入1%BSA稀释（0.01MPBS稀释）封闭液，37度恒温烘箱1h，顷去多余液体，不洗
7.滴加一抗用1%BSA稀释(1:50）或（1:100），先室温放置1h，然后4度过夜（放入湿盒）并用PBS代替一抗做阴性对照
8.PBS洗3次每次5min
9.滴加二抗（1:100）或（1:200），用1%BSA稀释37度恒温箱（放入湿盒）1h，避光
10.PBS洗3次各2min，避光，用摇床洗涤
11.加入20ul样品Hoechst液体（10ug/ml）5min用摇床避光
12.PBS洗3次各2min，避光，用摇床洗涤
13.滴加抗荧光猝灭剂，封片，镜检，绿色荧光
protocol如下：
灌注取脑后，4%多聚甲醛4℃固定24-48h。修块3-4mm。
脱水（常温）：45%、55%、65%、75%、85%、95%、100%酒精各30min；
透明（常温）：（100%酒精+二甲苯）/210min，二甲苯①20min，二甲苯②至完全透明（约20min）
浸蜡（60℃）：（二甲苯+石蜡①）/210min，石蜡①30min，石蜡②40min；石蜡①为低熔点，石蜡②为高熔点
包埋：浸蜡后用石蜡②手动包埋；
切片厚度：6um、（8um也切的有）
展片：40℃水中展片；
烤片：60摄氏度烤片机侧烤约5min后吸去水分，转移至60℃温箱烤约4h；
脱蜡：烤片后趁蜡未凝固，进行脱蜡。二甲苯5min×2，100%酒精3min，95%酒精2min，
85%酒精2min，75%酒精2min，流水冲洗2min（放在缸子里用水泡的，换了几次水）；
染色：
HE染色：苏木精5-8min（新配为5min，时间长可为8min，用前过滤），流水冲洗3min
1%盐酸酒精分化5s，流水冲洗1min，0.1%伊红1-2min，
脱水：85%酒精2min，95%酒精2min，100%酒精2min，
100%无水乙醇：二甲苯（1:1）2min
透明：二甲苯（I）3min，二甲苯（II）3min；
CV染色：浸入CV染液中10-20min，
脱水：75%乙醇2min，85%乙醇2min，95%乙醇2min，100%无水乙醇2min，
100%无水乙醇：二甲苯（1:1）2min
透明：二甲苯（I）3min，二甲苯（II）3min；
封片：中性树胶封片（整个染色过程避免干片）。
部分HE染色、CV染色片子结果如图所示，基本上没有完整的片子（染色时，片子随着在液体里的时间脱片、烂片的程度加深），另CV染色着色浅。
请教战友是什么原因？哪里出了问题，该怎么解决？
主药:50mg
mcc:20mg
l-hpc:10mg
cms-na:10mg
5%pvp50%乙醇qs
外加
ms:0.5mg
cms-na:3mg
50度干燥
以上处方不粘冲,但因上处方的辅料与主药有相互作用,导致片子变黄
现换成以下处方
处方1
主药:50mg
预胶化淀粉:20mg
淀粉:20mg
8%淀粉浆:qs
外加
ms:0.5或1mg
滑石粉:3或5mg
60度干燥
以上处方粘冲,为何?
处方2
主药:50mg
预胶化淀粉:20mg
淀粉:10mg
甘露醇:10mg
8%淀粉浆:qs
外加
ms:0.5或1mg
滑石粉:3或5mg
60度干燥
以上处方粘冲,为何
